Antisense modulation of connective tissue growth factor expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of connective tissue growth factor. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding connective tissue growth factor. Methods of using these compounds for modulation of connective tissue growth factor expression and for treatment of diseases associated with expression of connective tissue growth factor are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of connective tissue growth factor. Inparticular, this invention relates to compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodingconnective tissue growth factor. Such compounds have been shown tomodulate the expression of connective tissue growth factor.

BACKGROUND OF THE INVENTION

[0002] In the course of studies of platelet-derived growth factor (PDGF)isoforms, a novel, cysteine-rich mitogenic peptide secreted by humanvascular endothelial cells and related to the v-src-induced immediateearly gene product CEF-10 was identified. An anti-PDGF antibody was usedto screen a human umbilical vein endothelial cell (HUVEC) expressionlibrary, and the gene encoding this novel mitogen was named connectivetissue growth factor (CTGF). The connective tissue growth factor proteinwas shown to stimulate DNA synthesis and promote chemotaxis offibroblasts (Bradham et al., J. Cell Biol., 1991, 114, 1285-1294).

[0003] Connective tissue growth factor (CTGF; also known as ctgrofact,fibroblast inducible secreted protein, fisp-12, NOV2, insulin-likegrowth factor-binding protein-related protein 2, IGFBP-rP2, IGFBP-8,HBGF-0.8, Hcs24, and ecogenin) is a member of the CCN (CTGF/CYR61/NOV)family of modular proteins, named for the first family membersidentified, connective tissue growth factor, cysteine-rich (CYR61), andnephroblastoma overexpressed (NOV), but the family also includes theproteins ELM-1 (expressed in low-metastatic cells), WISP-3(Wnt-1-induced secreted protein), and COP-1 (WISP-2). CCN proteins havebeen found to be secreted, extracellular matrix-associated proteins thatregulate cellular processes such as adhesion, migration, mitogenesis,differentiation, survival, angiogenesis, atherosclerosis,chondrogenesis, wound healing, tumorigenesis, and vascular and fibroticdiseases like scleroderma (Lau and Lam, Exp. Cell Res., 1999, 248,44-57).

[0004] In most cases, a single 2.4-kilobase connective tissue growthfactor transcript has been reported in expression studies, although 3.5-and 7-kilobase transcripts have been reported in glioblastoma cells.Connective tissue growth factor is expressed in fibroblasts duringnormal differentiation processes that involve extracellular matrix (ECM)production and remodeling, such as embryogenesis and uterinedecidualization following implantation. Connective tissue growth factoris also frequently overexpressed in fibrotic skin disorders such assystemic sclerosis, localized skin sclerosis, keloids, scar tissue,eosinophilic fasciitis, nodular fasciitis, and Dupuytren's contracture.Connective tissue growth factor mRNA or protein levels are elevated infibrotic lesions of major organs and tissues including the liver,kidney, lung, cardiovascular system, pancreas, bowel, eye, and gingiva.In mammary, pancreatic and fibrohistiocytic tumors characterized bysignificant connective tissue involvement, connective tissue growthfactor is overexpressed in the stromal compartment. In many cases,connective tissue growth factor expression is linked spatially andtemporally to the profibrogenic cytokine transforming growth factor-beta(TGF-β) (Moussad and Brigstock, Mol. Genet. Metab., 2000, 71, 276-292).

[0005] Connective tissue growth factor has been mapped to humanchromosomal region 6q23.1, proximal to the c-myb gene, and chromosomalabnormalities involving this region have been associated with humantumors, such as Wilms' tumor (Martinerie et al., Oncogene, 1992, 7,2529-2534).

[0006] Tumors with significant fibrotic and vascular components exhibitincreased connective tissue growth factor expression, and connectivetissue growth factor may be involved in the pathogenesis of pediatricmyofibroblastic tumors. Of 12 pediatric tumors examined, all showedmoderate to intense connective tissue growth factor expression in tumorcells and/or endothelial cells of the associated vasculature (Kasaragodet al., Pediatr. Dev. Pathol., 2001, 4, 37-45).

[0007] Connective tissue growth factor mRNA is also specificallyupregulated in malignant human leukemic lymphoblasts from children withacute lymphoblastic leukemia (ALL) (Vorwerk et al., Br. J. Cancer, 2000,83, 756-760), and both mRNA and protein levels are upregulated byTGF-beta in Hs578T human breast cancer cells in a dose-dependent manner,indicating that connective tissue growth factor is an importantneuroendocrine factor and a critical downstream effector of TGF-beta(Yang et al., J. Clin. Endocrinol. Metab., 1998, 83, 2593-2596).

[0008] Based on a region of amino acid homology to insulin-like growthfactor (IGF) binding proteins (IGFBPs), connective tissue growth factorwas hypothesized to regulate cell growth through IGF. Recombinant humanconnective tissue growth factor was expressed in a baculoviral systemand demonstrated to bind to IGF in vitro with low affinity, and thus,connective tissue growth factor was identified as a member of the IGFBPsuperfamily, and was given the name IGFBP-8 (Kim et al., Proc. Natl.Acad. Sci. U. S. A., 1997, 94, 12981-12986).

[0009] The role of connective tissue growth factor has been investigatedin a transgenic mouse. Transgenic mice overproducing the connectivetissue growth factor protein under control of the collagen promotercould develop and their embryonic and neonatal growth were normal, butthey displayed dwarfism within a few months of birth, bone density wasdecreased compared with normal mice, male testes were much smaller thannormal and fertility was affected. These results indicate that theeffects of overexpression of connective tissue growth factor affectsendochondral ossification, and may also regulate embryonic developmentof the testes (Nakanishi et al., Biochem. Biophys. Res. Commun., 2001,281, 678-681).

[0010] In cultured 22-day fetal rat calvarial osteoblasts, cortisolstimulates transcription of connective tissue growth factor in a time-and dose-dependent manner, and cyclohexamide did not preclude thiseffect, indicating that this upregulation was not protein synthesisdependent. Glucocorticoids have complex effects on bone, some due todirect regulation of specific genes expressed by osteoblasts, and someindirect, mediated by locally produced growth factors or their bindingproteins. IGFs have important stimulatory effects on bone formation, butglucocorticoids inhibit expression of IGFs. Because connective tissuegrowth factor binds to IGF, its increased expression could modulate theeffect of cortisol on bone (Pereira et al., Am. J. Physiol. Endocrinol.Metab., 2000, 279, E570-576). connective tissue growth factor may beregulated not only at the level of transcription, but also byproteolytic degradation, but this varies with cell type. In large vesselbovine endothelial cells, cyclic AMP (cAMP) was found to increaseexpression of intact connective tissue growth factor protein byinhibiting degradation, whereas TGF-beta stimulated neither mRNA norprotein levels. In microvessel cells, TGF-beta stimulates an increase inconnective tissue growth factor mRNA and both TGF-beta and cAMPstimulated proteolytic degradation, and these differences may have aneffect on angiogenesis and wound healing (Boes et al., Endocrinology,1999, 140, 1575-1580).

[0011] Purified murine connective tissue growth factor promotes theadhesion of primary human dermal microvascular endothelial cells(HMVECs) and of platelets through integrin receptors α_(V)β₃ andα_(IIb)β₃, respectively, suggesting its involvement in cell adhesionsignaling, hemostasis and thrombosis (Babic et al., Mol. Cell Biol.,1999, 19, 2958-2966; Jedsadayanmata et al., J. Biol. Chem., 1999, 274,24321-24327). connective tissue growth factor also stimulates migrationof HMVECs in culture through an integrin receptor α_(V)β₃-dependentmechanism. Furthermore, connective tissue growth factor can promotesurvival of HMVECs plated onto laminin but deprived of growth factors, acondition that otherwise induces apoptosis. In vivo, connective tissuegrowth factor induces neovascularization in rat corneal micropocketimplants. Thus, connective tissue growth factor is an angiogenicinducer, and may play a role in adhesion, migration, and survival ofendothelial cells during blood vessel growth, perhaps by delivering anantiapoptotic signal via its interaction with integrin α_(V)β₃ (Babic etal., Mol. Cell Biol., 1999, 19, 2958-2966).

[0012] In contrast, connective tissue growth factor may negativelyregulate growth of human prostate cells. Connective tissue growth factorexpression is upregulated during senescence of normal human prostateepithelial cells (HPECs), and connective tissue growth factor isresponsive to growth regulators such as all-trans retinoic acid (atRA),supporting a growth-regulatory role of connective tissue growth factorin prostatic epithelium (Lopez-Bermejo et al., Endocrinology, 2000, 141,4072-4080).

[0013] Expansion of ECM with fibrosis occurs in many tissues as part ofthe end-organ complications of diabetes, and advanced glycosylation endproducts (AGE) are implicated as one causitive factor in diabetic tissuefibrosis. In addition to being a potent inducer of ECM synthesis andangiogenesis, Connective tissue growth factor is increased in tissuesfrom rodent models of diabetes. AGE treatement of primary cultures ofCRL-2097 and CRL-1474 nonfetal human dermal fibroblasts resulted in anincrease in steady state levels of connective tissue growth factor mRNAas well as protein levels in conditioned medium and cell-associatedconnective tissue growth factor, while other IGFBP-related proteins werenot upregulated by AGE. Thus, AGE upregulates the profibrotic andproangiogenic protein connective tissue growth factor, which may play arole in diabetic complications (Twigg et al., Endocrinology, 2001, 142,1760-1769).

[0014] In a murine lung fibrosis model, an increase in connective tissuegrowth factor mRNA expression is also induced by bleomycin, a known lungfibrogenic agent (Lasky et al., Am. J. Physiol., 1998, 275, L365-371),as well as in bronchoalveolar lavage cells from patients with idiopathicpulmonary fibrosis and pulmonary sarcoidosis, in comparison to healthynonsmoking control subjects, indicating that connective tissue growthfactor is involved in the fibroproliferative response to injury (Allenet al., Am. J. Respir. Cell Moll. Biol., 1999, 21, 693-700). Similarly,in an experimental model of proliferative glomerulonephritis, connectivetissue growth factor mRNA expression was strongly increased inextracapillary and mesangial proliferative lesions and in areas ofperiglomerular fibrosis. The early glomerular connective tissue growthfactor overexpression coincided with a striking upregulation of TGF-βproteins, and the kinetics of connective tissue growth factor expressionstrongly suggest a role in glomerular repair, possibly downstream ofTGF-beta in this model of transient renal injury (Ito et al., J. Am.Soc. Nephrol., 2001, 12, 472-484).

[0015] Disclosed and claimed in U.S. Pat. No. 5,876,730 is asubstantially pure or isolated polypeptide characterized as having anamino acid sequence corresponding to the carboxy terminal amino acids ofa connective tissue growth factor (CTGF) protein, wherein thepolypeptide has an amino acid sequence beginning at amino acid residue247 or 248 from the N-terminus of connective tissue growth factor, anisolated polynucleotide sequence encoding the connective tissue growthfactor polypeptide, a recombinant expression vector which contains saidpolynucleotide, a host cell containing said expression vector, and apharmaceutical composition comprising a therapeutically effective amountof connective tissue growth factor polypeptide in a pharmaceuticallyacceptable carrier. Antisense oligonucleotides are generally disclosed(Brigstock and Harding, 1999).

[0016] Disclosed and claimed in U.S. Pat. Nos. 5,783,187; 5,585,270;6,232,064; 6,150,101; 6,069,006 and PCT Publication WO 00/35936 are anisolated polynucleotide encoding the connective tissue growth factorpolypeptide, expression vectors, host cells stably transformed ortransfected with said vectors; an isolated polynucleotide comprising 5′untranslated regulatory nucleotide sequences isolated from upstream ofconnective tissue growth factor, wherein said untranslated regulatorynucleotide sequences comprises a transcriptional and translationalinitiation region and wherein said sequence is a TGF-beta responsiveelement; an isolated nucleic acid construct comprising a non-codingregulatory sequence isolated upstream from a connective tissue growthfactor (CTGF) gene, wherein said non-coding regulatory sequence isoperably associated with a nucleic acid sequence which expresses aprotein of interest or antisense RNA, wherein said nucleic acid sequenceis heterologous to said non-coding sequence; and a fragment ofconnective tissue growth factor (CTGF) polypeptide having the ability toinduce ECM synthesis, collagen synthesis and/or myofibroblastdifferentiation, comprising an amino acid sequence encoded by at leastexon 2 or exon 3 of said polypeptide. Further claimed is a method foridentifying a composition which affects TGF-beta-induced connectivetissue growth factor expression, and a method of diagnosing apathological state in a subject suspected of having a pathology selectedfrom the group consisting of fibrotic disease and atherosclerosis, themethod comprising obtaining a sample suspected of containing connectivetissue growth factor, whereby detecting a difference in the level ofconnective tissue growth factor in the sample from the subject ascompared to the level of connective tissue growth factor in the normalstandard sample is diagnostic of a pathology characterized by a cellproliferative disorder associated with connective tissue growth factorin the subject. Further claimed is a method for ameliorating a cellproliferative disorder associated with connective tissue growth factor,comprising administering to a subject having said disorder, at the siteof the disorder, a composition comprising a therapeutically effectiveamount of an antibody or fragment thereof that binds to connectivetissue growth factor, wherein said antibody or fragment thereof does notbind to PDGF. Antisense oligonucleotides are generally disclosed(Grotendorst, 2000; Grotendorst and Bradham, 2001; Grotendorst andBradham, 2000; Grotendorst and Bradham, 1996; Grotendorst and Bradham,1998; Grotendorst and Bradham, 2000).

[0017] Disclosed and claimed in PCT Publication WO 99/66959 is a devicefor promoting neuronal regeneration, comprising a gene activated matrixcomprising a biocompatible matrix and at least one neuronal therapeuticencoding agent having an operably linked promoter device, wherein theneuronal therapeutic encoding agent encodes an inhibitor of neuronalcell growth, and wherein the inhibitor of neuronal cell growth isselected from the group consisting of NFB42, TGF-beta, connective tissuegrowth factor (CTGF), and macrophage migration inhibitory factor (MIF),and wherein the neuronal therapeutic encoding agent is selected from thegroup consisting of a nucleic acid molecule, a vector, an antisensenucleic acid molecule and a ribozyme (Baird et al., 1999).

[0018] Disclosed and claimed in PCT Publication WO 00/27868 is asubstantially pure connective tissue growth factor polypeptide orfunctional fragments thereof, an isolated polynucleotide sequenceencoding said polypeptide, said polynucleotide sequence wherein T canalso be U, a nucleic acid sequence complementary to said polynucleotidesequence, and fragments of said sequences that are at least 15 bases inlength and that will hybridize to DNA which encodes the amino acidsequence of the connective tissue growth factor protein under moderateto highly stringent conditions. Further claimed is an expression vectorincluding said polynucleotide, a host cell stably transformed with saidvector, an antibody that binds to said polypeptide, and a method forproducing said polypeptide. Further claimed is a method for inhibitingthe expression of connective tissue growth factor in a cell comprisingcontacting the cell with a polynucleotide which binds to a targetnucleic acid in the cell, wherein the polynucleotide inhibits theexpression of connective tissue growth factor in the cell, wherein thepolynucleotide is an antisense polynucleotide, as well as a kit for thedetection of connective tissue growth factor expression comprising acarrier means being compartmentalized to receive one or more containers,comprising at least one container containing at least one antisenseoligonucleotide that binds to connective tissue growth factor (Schmidtet al., 2000).

[0019] Disclosed and claimed in PCT Publication WO 00/13706 is a methodfor treating or preventing fibrosis, the method comprising administeringto a subject in need an effective amount of an agent that modulates,regulates or inhibits the expression or activity of connective tissuegrowth factor or fragments thereof, and wherein the agent is anantibody, an antisense oligonucleotide, or a small molecule. The methodis directed to treating kidney fibrosis and associated renal disorders,in particular, complications associated with diabetes and hypertension(Riser and Denichili, 2000).

[0020] Disclosed and claimed in PCT Publication WO 01/29217 is anisolated nucleic acid molecule comprising a nucleic acid sequenceencoding a polypeptide comprising an amino acid sequence selected from agroup comprising NOV1, NOV2 (connective tissue growth factor), and NOV3,a mature form or variant of an amino acid sequence selected from saidgroup, as well as a nucleic acid molecule comprising a nucleic acidsequence encoding a polypeptide comprising an amino acid sequenceselected from said group as well as mature and variant forms orfragments of said polypeptides, and the complement of said nucleic acidmolecule. Antisense oligonucleotides are generally disclosed (Prayaga etal., 2001).

[0021] Currently, there are no known therapeutic agents whicheffectively inhibit the synthesis of connective tissue growth factor andto date, investigative strategies aimed at modulating connective tissuegrowth factor function have involved the use of sodium butyrate (NaB),function blocking antibodies and antisense oligonucleotides.

[0022] Dietary factors are believed to play an important role in boththe development and prevention of human cancers, including breastcarcinoma. The dietary micronutrient NaB is a major end product ofdigestion of dietary starch and fiber, and is a potent growth inhibitorthat initiates cell differentiation of many cell types in vitro. NaBexerts its biological effects, in part, as a histone deacetylaseinhibitor in mammary epithelial cells, induces apoptotic cell death inHs578T estrogen-non-responsive human breast cancer cells, and canactivate different genes involved in cell cycle arrest depending on celltype. NaB specifically upregulates the expression of connective tissuegrowth factor in a dose-dependent manner, stimulating an increase inboth mRNA and protein levels in both cancerous and non-cancerous mammarycells (Tsubaki et al., J. Endocrinol., 2001, 169, 97-110).

[0023] TGF-beta has the unique ability to stimulate growth of normalfibroblasts in soft agar, a property of transformed cells. Connectivetissue growth factor cannot induce this anchorage-independent growthnormal rat kidney (NRK) fibroblasts, but connective tissue growth factorsynthesis and action are essential for TGF-β-inducedanchorage-independence. Antibodies to connective tissue growth factorspecifically blocked TGF-beta-induced anchorage-independent growth, andNRK fibroblasts transformed with a construct expressing the connectivetissue growth factor gene in the antisense orientation were notresponsive to TGF-beta in the anchorage-independent growth assay(Kothapalli et al., Cell Growth. Differ., 1997, 8, 61-68). TheseCTGF-antisense expressing NRK cells were also used to show thatTGF-beta-stimulated collagen synthesis is mediated by connective tissuegrowth factor, indicating that connective tissue growth factor may be auseful target for antifibrotic therapies (Duncan et al., Faseb J., 1999,13, 1774-1786).

[0024] The 3′-untranslated region (UTR) of the human connective tissuegrowth factor cDNA bears several consensus sequences for regulatoryelements. When the 3′-UTR was fused downstream of a reporter gene, itwas found to act as a strong cis-acting repressive element, and theantisense 3′-UTR had a similar, but stronger effect. (Kubota et al.,FEBS Lett., 1999, 450, 84-88). Comparison of the human and mouseconnective tissue growth factor 3′-UTRs revealed a conserved smallsegment of 91 bases. This region was amplified by RT-PCR from NIH3T3mouse fibroblasts and used to make a chimeric fusion construct foranalysis of its repressive effects. The mouse connective tissue growthfactor 3′-UTR in either the sense or the antisense orientation had astrong repressive effect on transcription of the reporter gene,indicating an orientation independence of this regulatory element (Kondoet al., Biochem. Biophys. Res. Commun., 2000, 278, 119-124).

[0025] A phosphorothioate antisense oligonucleotide, 16 nucleotides inlength and targeted to the translation initiation start site, was usedto inhibit expression of connective tissue growth factor and suppressproliferation and migration of bovine aorta vascular endothelial cellsin culture (Shimo et al., J. Biochem. (Tokyo), 1998, 124, 130-140). Thisantisense oligonucleotide was also used to show that connective tissuegrowth factor induces apoptosis in MCF-7 human breast cancer cells andthat TGF-beta-induced apoptosis is mediated, in part, by connectivetissue growth factor (Hishikawa et al., J. Biol. Chem., 1999, 274,37461-37466). The same antisense oligonucleotide was also found toinhibit the TGF-beta-mediated activation of caspase 3 and thus toinhibit induction of TGF-beta-mediated apoptosis in human aortic smoothmuscle cells (HASC) (Hishikawa et al., Eur. J. Pharmacol., 1999, 385,287-290). This antisense oligonucleotide was also used to blockconnective tissue growth factor expression and demonstrate that highblood pressure upregulates expression of connective tissue growth factorin mesangial cells, which in turn enhances ECM protein production andinduces apoptosis, contributing to the remodeling of mesangium andultimately glomerulosclerosis (Hishikawa et al., J. Biol. Chem., 2001,276, 16797-16803).

[0026] Consequently, there remains a long felt need for additionalagents capable of effectively inhibiting connective tissue growth factorfunction.

[0027] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of connective tissue growthfactor expression.

[0028] The present invention provides compositions and methods formodulating connective tissue growth factor expression.

SUMMARY OF THE INVENTION

[0029] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding connective tissue growth factor, and which modulate theexpression of connective tissue growth factor. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofconnective tissue growth factor in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofconnective tissue growth factor by administering a therapeutically orprophylactically effective amount of one or more of the antisensecompounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding connective tissue growth factor,ultimately modulating the amount of connective tissue growth factorproduced. This is accomplished by providing antisense compounds whichspecifically hybridize with one or more nucleic acids encodingconnective tissue growth factor. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding connective tissue growthfactor” encompass DNA encoding connective tissue growth factor, RNA(including pre-mRNA and mRNA) transcribed from such DNA, and also cDNAderived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translation of protein from the RNA, splicing of the RNA to yield one ormore mRNA species, and catalytic activity which may be engaged in orfacilitated by the RNA. The overall effect of such interference withtarget nucleic acid function is modulation of the expression ofconnective tissue growth factor. In the context of the presentinvention, “modulation” means either an increase (stimulation) or adecrease (inhibition) in the expression of a gene. In the context of thepresent invention, inhibition is the preferred form of modulation ofgene expression and mRNA is a preferred target.

[0031] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding connective tissue growth factor. The targeting processalso includes determination of a site or sites within this gene for theantisense interaction to occur such that the desired effect, e.g.,detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding connective tissue growth factor, regardless of thesequence(s) of such codons.

[0032] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0033] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′ UTR), known in the art to refer to the portionof an mRNA in the 5′ direction from the translation initiation codon,and thus including nucleotides between the 5′ cap site and thetranslation initiation codon of an mRNA or corresponding nucleotides onthe gene, and the 3′ untranslated region (3′ UTR), known in the art torefer to the portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5′ cap region may also be apreferred target region.

[0034] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0035] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0036] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0037] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0038] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0039] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0040] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0041] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0042] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0043] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0044] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEDS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0045] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0046] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0047] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0048] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0049] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0050] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0051] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0052] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0053] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0054] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0055] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N (CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0056] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0057] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0058] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂-CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0059] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0060] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, which is commonly owned with theinstant application and also herein incorporated by reference.

[0061] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0062] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0063] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0064] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0065] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0066] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0067] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0068] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0069] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0070] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0071] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0072] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of connective tissue growth factor is treated byadministering antisense compounds in accordance with this invention. Thecompounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an antisense compound to asuitable pharmaceutically acceptable diluent or carrier. Use of theantisense compounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example.

[0073] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding connective tissue growth factor, enabling sandwich and otherassays to easily be constructed to exploit this fact. Hybridization ofthe antisense oligonucleotides of the invention with a nucleic acidencoding connective tissue growth factor can be detected by means knownin the art. Such means may include conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of connective tissue growth factor in a sample may also beprepared.

[0074] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0075] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0076] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. applications Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999) each of whichis incorporated herein by reference in their entirety.

[0077] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0078] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0079] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0080] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0081] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0082] Emulsions The compositions of the present invention may beprepared and formulated as emulsions. Emulsions are typicallyheterogenous systems of one liquid dispersed in another in the form ofdroplets usually exceeding 0.1 μm in diameter. (Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0083] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0084] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0085] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0086] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0087] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0088] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0089] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0090] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0091] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0092] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0093] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0094] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0095] Liposomes

[0096] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0097] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0098] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0099] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0100] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0101] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0102] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0103] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0104] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0105] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0106] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0107] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ I(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0108] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0109] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0110] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0111] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0112] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0113] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0114] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0115] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0116] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0117] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0118] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0119] Penetration Enhancers

[0120] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0121] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0122] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0123] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0124] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0125] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0126] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0127] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0128] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0129] Carriers

[0130] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0131] Excipients

[0132] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0133] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0134] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0135] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0136] Other Components

[0137] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0138] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0139] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0140] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0141] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0142] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0143] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-Alkoxy Amidites

[0144] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0145] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling VA or ChemGenes,Needham Mass.).

[0146] 2′-Fluoro Amidites

[0147] 2′-Fluorodeoxyadenosine Amidites

[0148] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′, 5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

[0149] 2′-Fluorodeoxyguanosine

[0150] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0151] 2′-Fluorouridine

[0152] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0153] 2′-Fluorodeoxycytidine

[0154] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0155] 2′-O-(2-Methoxyethyl) modified amidites

[0156] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

[0157] 2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0158] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.).

[0159] 2′-O-Methoxyethyl-5-methyluridine

[0160] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

[0161] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0162] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

[0163]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0164] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

[0165]3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0166] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

[0167] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0168] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

[0169]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0170] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

[0171]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0172]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L) Tetrazole diisopropylamine (7.1g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M) wereadded with stirring, under a nitrogen atmosphere. The resulting mixturewas stirred for 20 hours at room temperature (TLC showed the reaction tobe 95% complete). The reaction mixture was extracted with saturatedNaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueous washes wereback-extracted with CH₂Cl₂ (300 mL), and the extracts were combined,dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

[0173] 2′-O-(Aminooxyethyl) Nucleoside Amidites and2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

[0174] 2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0175] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

[0176] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0177] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

[0178]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0179] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

[0180]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0181]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

[0182]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0183]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%).

[0184]5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0185]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

[0186] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0187] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2 HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

[0188] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0189] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 ml) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

[0190]5-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0191] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

[0192] 2′-(Aminooxyethoxy) nucleoside amidites

[0193] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0194]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0195] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0196] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0197] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0198] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0199] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-, 2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

[0200] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine

[0201] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

[0202]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0203] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

[0204] Oligonucleotide Synthesis

[0205] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0206] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

[0207] Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0208] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0209] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0210] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0211] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0212] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0213] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0214] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0215] Oligonucleoside Synthesis

[0216] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0217] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0218] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0219] PNA Synthesis

[0220] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0221] Synthesis of Chimeric Oligonucleotides

[0222] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0223] [2′-O-Me]—[2′-deoxy]—[2′-O-Mel] Chimeric PhosphorothioateOligonucleotides

[0224] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 380B, asabove. oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNAportion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 51and 3′ wings. The standard synthesis cycle is modified by increasing thewait step after the delivery of tetrazole and base to 600 s repeatedfour times for RNA and twice for 2′-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample wasagain lyophilized to dryness. The pellet is resuspended in 1M TBAF inTHF for 24 hrs at room temperature to deprotect the 2′ positions. Thereaction is then quenched with 1M TEAA and the sample is then reduced to½ volume by rotovac before being desalted on a G25 size exclusioncolumn. The oligo recovered is then analyzed spectrophotometrically foryield and for purity by capillary electrophoresis and by massspectrometry.

[0225] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[0226] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)]chimeric phosphorothioate oligonucleotides were prepared as per theprocedure above for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[0227] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[0228] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0229] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0230] Oligonucleotide Isolation

[0231] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0232] Oligonucleotide Synthesis—96 Well Plate Format

[0233] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0234] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0235] Oligonucleotide Analysis—96 Well Plate Format

[0236] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0237] Cell Culture and Oligonucleotide Treatment

[0238] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 6 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0239] T-24 Cells:

[0240] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum ((Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0241] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0242] A549 Cells:

[0243] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, VA). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0244] NHDF Cells:

[0245] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0246] HEK Cells:

[0247] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0248] HuVEC Cells:

[0249] The human umbilical vein endothilial cell line HuVEC was obtainedfrom the American Type Culture Collection (Manassas, Va.). HuVEC cellswere routinely cultured in EBM (Clonetics Corporation Walkersville, Md.)supplemented with SingleQuots supplements (Clonetics Corporation,Walkersville, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence were maintained for up to 15passages. Cells were seeded into 96-well plates (Falcon-Primaria #3872)at a density of 10000 cells/well for use in RT-PCR analysis.

[0250] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0251] b.END Cells:

[0252] The mouse brain endothelial cell line b.END was obtained from Dr.Werner Risau at the Max Plank Instititute (Bad Nauheim, Germany). b.ENDcells were routinely cultured in DMEM, high glucose (Gibco/LifeTechnologies, Gaithersburg, MD) supplemented with 10% fetal calf serum(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 3000 cells/well for use in RT-PCR analysis.

[0253] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0254] Treatment with Antisense Compounds:

[0255] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0256] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

[0257] Analysis of Oligonucleotide Inhibition of Connective TissueGrowth Factor Expression

[0258] Antisense modulation of connective tissue growth factorexpression can be assayed in a variety of ways known in the art. Forexample, connective tissue growth factor mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. The preferred method of RNA analysis of the presentinvention is the use of total cellular RNA as described in otherexamples herein. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRISM™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

[0259] Protein levels of connective tissue growth factor can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS). Antibodies directed toconnective tissue growth factor can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

[0260] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11

[0261] Poly(A)+mRNA Isolation

[0262] Poly(A)+mRNA was isolated according to Miura et al., Clin. Chem.,1996, 42, 1758-1764. Other methods for poly(A)+mRNA isolation are taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

[0263] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0264] Total RNA Isolation

[0265] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0266] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0267] Real-Time Quantitative PCR Analysis of Connective Tissue GrowthFactor mRNA Levels

[0268] Quantitation of connective tissue growth factor mRNA levels wasdetermined by real-time quantitative PCR using the ABI PRISMS 7700Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR, in which amplificationproducts are quantitated after the PCR is completed, products inreal-time quantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM, obtainedfrom either Operon Technologies Inc., Alameda, Calif. or Integrated DNATechnologies Inc., Coralville, Iowa.) is attached to the 5′ end of theprobe and a quencher dye (e.g., TAMRA, obtained from either OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa.) is attached to the 3′ end of the probe. When theprobe and dyes are intact, reporter dye emission is quenched by theproximity of the 3′ quencher dye. During amplification, annealing of theprobe to the target sequence creates a substrate that can be cleaved bythe 5′-exonuclease activity of Taq polymerase. During the extensionphase of the PCR amplification cycle, cleavage of the probe by Taqpolymerase releases the reporter dye from the remainder of the probe(and hence from the quencher moiety) and a sequence-specific fluorescentsignal is generated. With each cycle, additional reporter dye moleculesare cleaved from their respective probes, and the fluorescence intensityis monitored at regular intervals by laser optics built into the ABIPRISM™ 7700 Sequence Detection System. In each assay, a series ofparallel reactions containing serial dilutions of mRNA from untreatedcontrol samples generates a standard curve that is used to quantitatethe percent inhibition after antisense oligonucleotide treatment of testsamples.

[0269] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0270] PCR reagents were obtained from Invitrogen, Carlsbad, Calif.RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCRbuffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP,375 nM each of forward primer and reverse primer, 125 nM of probe, 4Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reversetranscriptase, and 2.5×ROX dye) to 96 well plates containing 30 μL totalRNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0271] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,Analytical Biochemistry, 1998, 265, 368-374.

[0272] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0273] Probes and primers to human connective tissue growth factor weredesigned to hybridize to a human connective tissue growth factorsequence, using published sequence information (GenBank accession numberM92934.1, incorporated herein as SEQ ID NO: 3). For human connectivetissue growth factor the PCR primers were: forward primer:ACAAGGGCCTCTTCTGTGACTT (SEQ ID NO: 4) reverse primer:GGTACACCGTACCACCGAAGAT (SEQ ID NO: 5) and the PCR probe was:FAM-TGTGCACCGCCAAAGATGGTGCT-TAMRA (SEQ ID NO: 6) where FAM (PE-AppliedBiosystems, Foster City, Calif.) is the fluorescent reporter dye) andTAMRA (PE-Applied Biosystems, Foster City, Calif.) is the quencher dye.For human GAPDH the PCR primers were: forward primer:GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 7) reverse primer: GAAGATGGTGATGGGATTTC(SEQ ID NO: 8) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA3′ (SEQ ID NO: 9) where JOE (PE-Applied Biosystems, Foster City, Calif.)is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,Foster City, Calif.) is the quencher dye.

[0274] Probes and primers to mouse connective tissue growth factor weredesigned to hybridize to a mouse connective tissue growth factorsequence, using published sequence information (GenBank accession numberBC006783.1, incorporated herein as SEQ ID NO: 10). For mouse connectivetissue growth factor the PCR primers were: forward primer:GGCAAATTCAACGGCACAGT (SEQ ID NO: 11) reverse primer: GCCCCCCACCCCAAA(SEQ ID NO: 12) and the PCR probe was:FAM-TCATAATCAAAGAAGCAGCAAGCACTTCCTG-TAMRA (SEQ ID NO: 13) where FAM(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye. For mouse GAPDH the PCR primers were: forward primer:GGCAAATTCAACGGCACAGT(SEQ ID NO: 14) reverse primer:GGGTCTCGCTCCTGGAAGAT(SEQ ID NO: 15) and the PCR probe was: 5′JOE-AAGGCCGAGAATGGGAAGCTTGTCATC-TAMRA 3′ (SEQ ID NO: 16) where JOE(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent reporterdye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) is thequencher dye.

Example 14

[0275] Northern Blot Analysis of Connective Tissue Growth Factor mRNALevels

[0276] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio.).RNA was transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0277] To detect human connective tissue growth factor, a humanconnective tissue growth factor specific probe was prepared by PCR usingthe forward primer ACAAGGGCCTCTTCTGTGACTT (SEQ ID NO: 4) and the reverseprimer GGTACACCGTACCACCGAAGAT (SEQ ID NO: 5). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0278] To detect mouse connective tissue growth factor, a mouseconnective tissue growth factor specific probe was prepared by PCR usingthe forward primer GCTCAGGGTAAGGTCCGATTC (SEQ ID NO: 11) and the reverseprimer GCCCCCCACCCCAAA (SEQ ID NO: 12). To normalize for variations inloading and transfer efficiency membranes were stripped and probed formouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,Palo Alto, Calif.).

[0279] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0280] Antisense Inhibition of Human Connective Tissue Growth FactorExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap

[0281] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanconnective tissue growth factor RNA, using published sequences (GenBankaccession number M92934.1, incorporated herein as SEQ ID NO: 3, GenBankaccession number X78947.1, incorporated herein as SEQ ID NO: 17, GenBankaccession number XM_(—)037055.1, incorporated herein as SEQ ID NO: 18,and GenBank accession number XM_(—)037056.1, incorporated herein as SEQID NO: 19). The oligonucleotides are shown in Table 1. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the oligonucleotide binds. All compounds in Table 1are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human connective tissue growth factor mRNA levels byquantitative real-time PCR as described in other examples herein. Dataare averages from two experiments. If present, “N.D.” indicates “nodata”. TABLE 1 Inhibition of human connective tissue growth factor mRNAlevels by chimeric phosphorothioate oligonucleotides having 2′-MOE wingsand a deoxy gap TARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITESEQUENCE % INHIB NO 100880 Coding 3 707 gcagttggctctaatcatag 0 20 100883Coding 3 828 tgaccatgcacaggcggctc 12 21 100885 Coding 3 917ctcaaacttgataggcttgg 18 22 100886 Coding 3 956 tttagctcggtatgtcttca 0 23100888 Coding 3 1028 cttgaactccaccggcaggg 28 24 100889 Coding 3 1076ggtcttgatgaacatcatgt 40 25 100890 Coding 3 1098 gacagttgtaatggcaggca 2326 100891 Coding 3 1147 ccgtacatcttcctgtagta 31 27 124173 Coding 18 304ccagctgcttggcgcagacg 80 28 124183 Coding 18 718 tctggaccaggcagttggct 1229 124184 Coding 18 723 tgtggtctggaccaggcagt 1 30 124185 Coding 18 728cactctgtggtctggaccag 16 31 124188 Coding 18 882 gatgcactttttgcccttct 032 124189 Coding 18 927 gccagaaagctcaaacttga 77 33 124190 Coding 18 932gtgcagccagaaagctcaaa 37 34 124196 Coding 18 1079 caggtcttgatgaacatcat 3335 124197 Coding 18 1084 aggcacaggtcttgatgaac 53 36 124198 Coding 181089 atggcaggcacaggtcttga 9 37 124199 Coding 18 1098acagttgtaatggcaggcac 72 38 124212 3′UTR 18 1707 ccacaagctgtccagtctaa 6639 124213 3′UTR 18 1712 acttgccacaagctgtccag 1 40 124215 3′UTR 18 1815ttaacttagataactgtaca 79 41 124216 3′UTR 18 1820 ttaaattaacttagataact 042 124230 3′UTR 19 2098 ttaataaaggccatttgttc 0 43 124234 3′UTR 19 2198cactctcaacaaataaactg 14 44 124235 3′UTR 19 2203 ggtcacactctcaacaaata 8745 124236 3′UTR 19 2208 cttttggtcacactctcaac 35 46 124237 3′UTR 19 2213tgtaacttttggtcacactc 35 47 124238 3′UTR 19 2218 aaacatgtaacttttggtca 8948 124239 3′UTP 19 2242 ctttattttcaactagaaag 0 49 144294 Coding 18 303cagctgcttggcgcagacgc 30 50 144305 Coding 18 622 ccttgggctcgtcacacacc 051 144311 Coding 18 725 tctgtggtctggaccaggca 49 52 144314 Coding 18 929cagccagaaagctcaaactt 0 53 144315 Coding 18 935 ctggtgcagccagaaagctc 0 54144319 Coding 18 1080 acaggtcttgatgaacatca 0 55 144320 Coding 18 1086gcaggcacaqgtcttgatga 0 56 144321 Coding 18 1091 taatggcaggcacaggtctt 057 144323 Coding 18 1156 ccatgtctccgtacatcttc 28 58 144336 3′UTP 18 1711cttgccacaagctgtccagt 1 59 144337 3′UTR 18 1740 aaaaatctggcttgttacag 0 60144338 3′UTR 18 1822 ctttaaattaacttagataa 0 61 144345 3′UTR 19 2206tttggtcacactctcaacaa 38 62 144346 3′UTR 19 2212 gtaacttttggtcacactct 3663 144347 3′UTR 19 2219 caaacatgtaacttttggtc 24 64 144348 3′UTR 19 2243actttattttcaactagaaa 0 65 144802 Start 3 120 cggcggtcatggttggcact 0 66Codon 144803 Start 3 130 cccatactggcggcggtcat 0 67 144804 Coding 17 284ccgtccagcacgaggctcac 49 68 144805 Coding 17 367 agaggcccttgtgcgggtcg 069 144806 Coding 17 383 gagccgaagtcacagaagag 68 70 144807 Coding 17 473aaggactctccgctgcggta 0 71 144808 Coding 3 487 cacgtgcactggtacttgca 45 72144809 Coding 17 611 tcgcagcatttcccgggcag 73 73 144810 Coding 17 615ctcctcgcagcatttcccgg 7 74 144811 Coding 17 633 gggctcgtcacacacccact 1775 144812 Coding 17 699 gtctgggccaaacgtgtctt 0 76 144813 Coding 3 698tctaatcatagttgggtctg 6 77 144814 Coding 17 729 gaccaggcagttggctctaa 0 78144815 Coding 17 819 ctctagcctgoaggaggcgt 0 79 144816 Coding 17 875atgttctcttccaggtcagc 0 80 144817 Coding 17 915 ggagattttgggagtacgga 5181 144818 Coding 17 926 ttgataggcttggagatttt 1 82 144820 Coding 17 979cacagaatttagctcggtat 0 83 144822 Coding 3 981 ggccgtcggtacatactcca 0 84144824 Coding 17 1037 tccaocggcagggtggtggt 16 85 144826 Coding 17 1051cagggcacttgaactccacc 41 86 144828 Coding 17 1055 ccgtcagggcacttgaactc 087 144830 Coding 17 1115 ggacagttgtaatggcaggc 39 88 144833 Coding 171149 gtagtacagcgattcaaaga 13 89 144835 Stop 3 1167 tctggcttcatgccatgtct37 90 Codon 144837 Stop 3 1177 tctctcactctctggcttca 25 91 Codon 1448393′UTR 3 1229 tacggaaaaatgagatgtga 41 92 144841 3′UTR 3 1261atttaaataacttgtgctac 0 93 144843 3′UTR 3 1358 ttcttcaaaccagtgtctgg 0 94144845 3′UTR 3 1537 cagtgagcacgctaaaattt 72 95 144847 3′UTR 3 1621gttctgacttaaggaacaac 0 96 144849 3′UTR 3 1697 gctgtccagtctaatcgaca 52 97

[0282] As shown in Table 1, SEQ ID NOs 24, 25, 27, 28, 33, 34, 35, 36,38, 39, 41, 45, 46, 47, 48, 50, 52, 58, 62, 63, 64, 68, 70, 72, 73, 81,86, 88, 90, 91, 92, 95 and 97 demonstrated at least 24% inhibition ofhuman connective tissue growth factor expression in this assay and aretherefore preferred. The target sites to which these preferred sequencesare complementary are herein referred to as “active sites” and aretherefore preferred sites for targeting by compounds of the presentinvention.

Example 16

[0283] Antisense Inhibition of Mouse Connective Tissue Growth FactorExpression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOEWings and a Deoxy Gap.

[0284] In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseconnective tissue growth factor RNA, using published sequences (GenBankaccession number BC006783.1, incorporated herein as SEQ ID NO: 10, andGenBank accession number M80263.1, incorporated herein as SEQ ID NO:98). The oligonucleotides are shown in Table 2. “Target site” indicatesthe first (5′-most) nucleotide number on the particular target sequenceto which the oligonucleotide binds. All compounds in Table 2 arechimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composedof a central “gap” region consisting of ten 2′-deoxynucleotides, whichis flanked on both sides (5′ and 3′ directions) by five-nucleotide“wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides.The internucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on mouseconnective tissue growth factor mRNA levels by quantitative real-timePCR as described in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”. TABLE 2 Inhibitionof mouse connective tissue growth factor mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET SEQ ID ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB NO100891 Coding 10 1220 ccgtacatcttcctgtagta 78 27 124173 Coding 98 374ccagctgcttggcgcagacg 15 28 124183 Coding 98 788 tctggaccaggcagttggct 2329 124184 Coding 98 793 tgtggtctggaccaggcagt 24 30 124185 Coding 98 798cactctgtggtctggaccag 41 31 124188 Coding 98 952 gatgcactttttgcccttct 2532 124189 Coding 98 997 gccagaaagctcaaacttga 79 33 124190 Coding 98 1002gtgcagccagaaagctcaaa 72 34 124196 Coding 98 1149 caggtcttgatgaacatcat 1235 124197 Coding 98 1154 aggcacaggtcttgatgaac 0 36 124198 Coding 98 1159atggcaggcacaggtcttga 32 37 124199 Coding 98 1168 acagttgtaatggcaggcac 3738 124212 3′UTR 98 1774 ccacaagctgtccagtctaa 80 39 124213 3′UTR 98 1779acttgccacaagctgtccag 52 40 124215 3′UTR 98 1874 ttaacttagataactgtaca 5441 124216 3′UTR 98 1879 ttaaattaacttagataact 24 42 124230 3′UTR 98 2131ttaataaaggccatttgttc 3 43 124234 3′UTR 98 2235 cactctcaacaaataaactg 1844 142353 3′UTP 98 2240 ggtcacactctcaacaaata 16 45 124236 3′UTR 98 2245cttttggtcacactctcaac 0 46 124237 3′UTR 98 2250 tgtaacttttggtcacactc 5747 124238 3′UTR 98 2255 aaacatgtaacttttggtca 36 48 124239 3′UTR 98 2278ctttattttcaactagaaag 0 49 100884 Coding 10 949 actttttgcccttcttaatg 1499 124165 5′UTR 98 88 gacgctccaggcggtggcgt 0 100 124166 5′UTR 98 93gtctggacgctccaggcggt 0 101 124167 5′UTR 98 131 cggctggagcctggattcgg 23102 124168 5′UTR 98 139 gagaggcgcggctggagcct 43 103 124169 5′UTR 98 177acgcggtaggaggatgcaca 24 104 124170 Start 98 195 gaggcgagcatgatcgggac 0105 Codon 124171 Coding 98 364 ggcgcagacgcggcagcagc 68 106 124172 Coding98 369 tgcttggcgcagacgcggca 0 107 124174 Coding 98 418gcccttgtgtgggtcgcagg 42 108 124175 Coding 98 431 aatcgcagaagaggcccttg 1109 124176 Coding 98 507 accgacccaccgaagacaca 45 110 124177 Coding 98550 ttggtatttgcagctgcttt 34 111 124178 Coding 98 583cacgcagcccacggccccat 0 112 124179 Coding 98 605 gcacgtccatgctgcatagg 18113 124180 Coding 98 650 gcagcttgacccttctcggg 20 114 124181 Coding 98705 actgctgtgcggtccttggg 12 115 124182 Coding 98 741gtgtcttccagtcggtaggc 60 116 124186 Coding 98 861 aaggtattgtcattggtaac 37117 124187 Coding 98 884 ggctctgcttctccagtctg 5 118 124191 Coding 981013 tcttcacactggtgcagcca 0 119 124192 Coding 98 1049cgtctgtgcacaccccgcag 31 120 124193 Coding 10 1068 cggtgtgcagcagcggccgt 5121 124194 Coding 98 1092 tccactggcagagtggtggt 47 122 124195 Coding 981135 catcatattctttttcatga 1 123 124200 Coding 98 1183gtcattgtccccaggacagt 33 124 124201 Stop 98 1239 tcctggctttacgccatgtc 13125 Codon 124202 3′UTR 98 1293 aaatgagatgcaactcagtt 28 126 124203 3′UTR98 1487 tcagtgtgcgttctggcact 38 127 124204 3′UTR 98 1504gttccaggagactcacctca 54 128 124205 3′UTR 98 1512 tctccactgttccaggagac 1129 124206 3′UTR 98 1522 tctcctggcatctccactgt 38 130 124207 3′UTR 981528 tttctttctcctggcatctc 0 131 124208 3′UTR 98 1594tccccggttacactccaaaa 0 132 124209 3′UTR 98 1625 aggtctgtctgcaagcatgc 0133 124210 3′UTR 98 1645 tgctcagctctcgctagagc 45 134 124211 3′UTR 981730 agtgtcactggaatcagaat 47 135 124214 3′UTR 98 1856caaatatatatatatatata 42 136 124217 3′UTR 98 1902 acttaaaacaaaaacaaatg 0137 124218 3′UTR 98 1927 gctatcagtttaaaatccca 42 138 124219 3′UTR 981957 gtgtoctacctatggtgttt 48 139 124220 3′UTR 98 1978tttgaatcacagataagctt 48 140 124221 3′UTR 98 1993 cagtatctcctttgttttga 34141 124222 3′UTR 98 2003 attcccactgcagtatctcc 44 142 124223 3′UTR 982012 caggtcacaattcccactgc 30 143 124224 3′UTR 98 2028ctgacagagagtcactcagg 40 144 124225 3′UTR 98 2058 gctttatcacctgcacagca 74145 124226 3′UTR 98 2064 tacatagctttatcacctgc 56 146 124227 3′UTR 982071 cttccaatacatagctttat 66 147 124228 3′UTR 98 2076tctgacttccaatacatagc 18 148 124229 3′UTR 98 2119 atttgttcaccaacagggat 0149 124231 3′UTR 98 2152 ttaccctgagccagccattt 14 150 124232 3′UTR 982188 aagaagcagcaagcacttcc 40 151 124233 3′UTR 98 2200cagtcataatcaaagaagca 1 152 124240 3′UTR 98 2283 atatactttattttcaacta 51153

[0285] As shown in Table 2, SEQ ID NOs 27, 30, 31, 32, 33, 34, 37, 39,40, 41, 42, 47, 48, 103, 104, 106, 108, 110, 111, 116, 117, 120, 122,124, 126, 127, 128, 130, 134, 135, 136, 138, 139, 140, 141, 142, 143,144, 145, 146, 147, 151 and 153 demonstrated at least 24% inhibition ofmouse connective tissue growth factor expression in this experiment andare therefore preferred. The target sites to which these preferredsequences are complementary are herein referred to as “active sites” andare therefore preferred sites for targeting by compounds of the presentinvention.

Example 17

[0286] Western Blot Analysis of Connective Tissue Growth Factor ProteinLevels

[0287] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to connective tissuegrowth factor is used, with a radiolabelled or fluorescently labeledsecondary antibody directed against the primary antibody species. Bandsare visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

1 153 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 atgcattctg cccccaagga 20 3 2075 DNA Homo sapiens CDS(130)...(1179) 3 cccggccgac agccccgaga cgacagcccg gcgcgtcccg gtccccacctccgaccaccg 60 ccagcgctcc aggccccgcg ctccccgctc gccgccaccg cgccctccgctccgcccgca 120 gtgccaacc atg acc gcc gcc agt atg ggc ccc gtc cgc gtc gccttc gtg 171 Met Thr Ala Ala Ser Met Gly Pro Val Arg Val Ala Phe Val 1 510 gtc ctc ctc gcc ctc tgc agc cgg ccg gcc gtc ggc cag aac tgc agc 219Val Leu Leu Ala Leu Cys Ser Arg Pro Ala Val Gly Gln Asn Cys Ser 15 20 2530 ggg ccg tgc cgg tgc ccg gac gag ccg gcg ccg cgc tgc ccg gcg ggc 267Gly Pro Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys Pro Ala Gly 35 40 45gtg agc ctc gtg ctg gac ggc tgc ggc tgc tgc cgc gtc tgc gcc aag 315 ValSer Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val Cys Ala Lys 50 55 60 cagctg ggc gag ctg tgc acc gag cgc gac ccc tgc gac ccg cac aag 363 Gln LeuGly Glu Leu Cys Thr Glu Arg Asp Pro Cys Asp Pro His Lys 65 70 75 ggc ctcttc tgt gac ttc ggc tcc ccg gcc aac cgc aag atc ggc gtg 411 Gly Leu PheCys Asp Phe Gly Ser Pro Ala Asn Arg Lys Ile Gly Val 80 85 90 tgc acc gccaaa gat ggt gct ccc tgc atc ttc ggt ggt acg gtg tac 459 Cys Thr Ala LysAsp Gly Ala Pro Cys Ile Phe Gly Gly Thr Val Tyr 95 100 105 110 cgc agcgga gag tcc ttc cag agc agc tgc aag tac cag tgc acg tgc 507 Arg Ser GlyGlu Ser Phe Gln Ser Ser Cys Lys Tyr Gln Cys Thr Cys 115 120 125 ctg gacggg gcg gtg ggc tgc atg ccc ctg tgc agc atg gac gtt cgt 555 Leu Asp GlyAla Val Gly Cys Met Pro Leu Cys Ser Met Asp Val Arg 130 135 140 ctg cccagc cct gac tgc ccc ttc ccg agg agg gtc aag ctg ccc ggg 603 Leu Pro SerPro Asp Cys Pro Phe Pro Arg Arg Val Lys Leu Pro Gly 145 150 155 aaa tgctgc gag gag tgg gtg tgt gac gag ccc aag gac caa acc gtg 651 Lys Cys CysGlu Glu Trp Val Cys Asp Glu Pro Lys Asp Gln Thr Val 160 165 170 gtt gggcct gcc ctc gcg gct tac cga ctg gaa gac acg ttt ggc cca 699 Val Gly ProAla Leu Ala Ala Tyr Arg Leu Glu Asp Thr Phe Gly Pro 175 180 185 190 gaccca act atg att aga gcc aac tgc ctg gtc cag acc aca gag tgg 747 Asp ProThr Met Ile Arg Ala Asn Cys Leu Val Gln Thr Thr Glu Trp 195 200 205 agcgcc tgt tcc aag acc tgt ggg atg ggc atc tcc acc cgg gtt acc 795 Ser AlaCys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr Arg Val Thr 210 215 220 aatgac aac gcc tcc tgc agg cta gag aag cag agc cgc ctg tgc atg 843 Asn AspAsn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg Leu Cys Met 225 230 235 gtcagg cct tgc gaa gct gac ctg gaa gag aac att aag aag ggc aaa 891 Val ArgPro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys Lys Gly Lys 240 245 250 aagtgc atc cgt act ccc aaa atc tcc aag cct atc aag ttt gag ctt 939 Lys CysIle Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys Phe Glu Leu 255 260 265 270tct ggc tgc acc agc atg aag aca tac cga gct aaa ttc tgt gga gta 987 SerGly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe Cys Gly Val 275 280 285tgt acc gac ggc cga tgc tgc acc ccc cac aga acc acc acc ctg ccg 1035 CysThr Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr Thr Leu Pro 290 295 300gtg gag ttc aag tgc cct gac ggc gag gtc atg aag aag aac atg atg 1083 ValGlu Phe Lys Cys Pro Asp Gly Glu Val Met Lys Lys Asn Met Met 305 310 315ttc atc aag acc tgt gcc tgc cat tac aac tgt ccc gga gac aat gac 1131 PheIle Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly Asp Asn Asp 320 325 330atc ttt gaa tcg ctg tac tac agg aag atg tac gga gac atg gca tga 1179 IlePhe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp Met Ala * 335 340 345agccagagag tgagagacat taactcatta gactggaact tgaactgatt cacatctcat 1239ttttccgtaa aaatgatttc agtagcacaa gttatttaaa tctgtttttc taactggggg 1299aaaagattcc cacccaattc aaaacattgt gccatgtcaa acaaatagtc tatcttcccc 1359agacactggt ttgaagaatg ttaagacttg acagtggaac tacattagta cacagcacca 1419gaatgtatat taaggtgtgg ctttaggagc agtgggaggg taccggcccg gttagtatca 1479tcagatcgac tcttatacga gtaatatgcc tgctatttga agtgtaattg agaaggaaaa 1539ttttagcgtg ctcactgacc tgcctgtagc cccagtgaca gctaggatgt gcattctcca 1599gccatcaaga gactgagtca agttgttcct taagtcagaa cagcagactc agctctgaca 1659ttctgattcg aatgacactg ttcaggaatc ggaatcctgt cgattagact ggacagcttg 1719tggcaagtga atttgcctgt aacaagccag attttttaaa atttatattg taaatattgt 1779gtgtgtgtgt gtgtgtgtat atatatatat atatgtacag ttatctaagt taatttaaag 1839ttgtttgtgc ctttttattt ttgtttttaa tgctttgata tttcaatgtt agcctcaatt 1899tctgaacacc ataggtagaa tgtaaagctt gtctgatcgt tcaaagcatg aaatggatac 1959ttatatggaa attctgctca gatagaatga cagtccgtca aaacagattg tttgcaaagg 2019ggaggcatca gtgtcttggc aggctgattt ctaggtagga aatgtggtag ctcacg 2075 4 22DNA Artificial Sequence PCR Primer 4 acaagggcct cttctgtgac tt 22 5 22DNA Artificial Sequence PCR Primer 5 ggtacaccgt accaccgaag at 22 6 23DNA Artificial Sequence PCR Probe 6 tgtgcaccgc caaagatggt gct 23 7 19DNA Artificial Sequence PCR Primer 7 gaaggtgaag gtcggagtc 19 8 20 DNAArtificial Sequence PCR Primer 8 gaagatggtg atgggatttc 20 9 20 DNAArtificial Sequence PCR Probe 9 caagcttccc gttctcagcc 20 10 2334 DNA Musmusculus CDS (206)...(1252) 10 gaagactcag ccagatccac tccagctccgaccccaggag accgacctcc tccagacggc 60 agcagcccca gcccagccga caaccccagacgccaccgcc tggagcgtcc agacaccaac 120 ctccgcccct gtccgaatcc aggctccggccgcgcctctc gtcgcctctg caccctgctg 180 tgcatcctcc taccgcgtcc cgatc atg ctcgcc tcc gtc gca ggt ccc atc 232 Met Leu Ala Ser Val Ala Gly Pro Ile 1 5agc ctc gcc ttg gtg ctc ctc gcc ctc tgc acc cgg cct gct acg ggc 280 SerLeu Ala Leu Val Leu Leu Ala Leu Cys Thr Arg Pro Ala Thr Gly 10 15 20 25cag gac tgc agc gcg caa tgt cag tgc gca gcc gaa gca gcg ccg cac 328 GlnAsp Cys Ser Ala Gln Cys Gln Cys Ala Ala Glu Ala Ala Pro His 30 35 40 tgcccc gcc ggc gtg agc ctg gtg ctg gac ggc tgc ggc tgc tgc cgc 376 Cys ProAla Gly Val Ser Leu Val Leu Asp Gly Cys Gly Cys Cys Arg 45 50 55 gtc tgcgcc aag cag ctg gga gaa ctg tgt acg gag cgt gac ccc tgc 424 Val Cys AlaLys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys 60 65 70 gac cca cacaag ggc ctc ttc tgc gat ttc ggc tcc ccc gcc aac cgc 472 Asp Pro His LysGly Leu Phe Cys Asp Phe Gly Ser Pro Ala Asn Arg 75 80 85 aag atc gga gtgtgc act gcc aaa gat ggt gca ccc tgt gtc ttc ggt 520 Lys Ile Gly Val CysThr Ala Lys Asp Gly Ala Pro Cys Val Phe Gly 90 95 100 105 ggg tcg gtgtac cgc agc ggt gag tcc ttc caa agc agc tgc aaa tac 568 Gly Ser Val TyrArg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr 110 115 120 caa tgc acttgc ctg gat ggg gcc gtg ggc tgc gtg ccc ctg tgc agc 616 Gln Cys Thr CysLeu Asp Gly Ala Val Gly Cys Val Pro Leu Cys Ser 125 130 135 atg gac gtgcgc ctg ccc agc cct gac tgc ccc ttc ccg aga agg gtc 664 Met Asp Val ArgLeu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val 140 145 150 aag ctg cctggg aaa tgc tgc gag gag tgg gtg tgt gac gag ccc aag 712 Lys Leu Pro GlyLys Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Lys 155 160 165 gac cgc acagca gtt ggc cct gcc cta gct gcc tac cga ctg gaa gac 760 Asp Arg Thr AlaVal Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp 170 175 180 185 aca tttggc cca gac cca act atg atg cga gcc aac tgc ctg gtc cag 808 Thr Phe GlyPro Asp Pro Thr Met Met Arg Ala Asn Cys Leu Val Gln 190 195 200 acc acagag tgg agc gcc tgt tct aag acc tgt ggg atg ggc atc tcc 856 Thr Thr GluTrp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile Ser 205 210 215 acc cgagtt acc aat gac aat acc ttc tgc aga ctt gag aag cag agt 904 Thr Arg ValThr Asn Asp Asn Thr Phe Cys Arg Leu Glu Lys Gln Ser 220 225 230 cgc ctctgc atg gtc agg ccc tgc gaa gct gac ctg gag gaa aac att 952 Arg Leu CysMet Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile 235 240 245 aag aagggc aaa aag tgc atc cgg aca cct aaa atc gcc aag cct gtc 1000 Lys Lys GlyLys Lys Cys Ile Arg Thr Pro Lys Ile Ala Lys Pro Val 250 255 260 265 aagttt gag ctt tct ggc tgc acc agt gtg aag aca tac agg gct aag 1048 Lys PheGlu Leu Ser Gly Cys Thr Ser Val Lys Thr Tyr Arg Ala Lys 270 275 280 ttctgc ggg gtg tgc aca gac ggc cgc tgc tgc aca ccg cac aga acc 1096 Phe CysGly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr 285 290 295 accact ctg cca gtg gag ttc aaa tgc ccc gat ggc gag atc atg aaa 1144 Thr ThrLeu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Ile Met Lys 300 305 310 aagaat atg atg ttc atc aag acc tgt gcc tgc cat tac aac tgt cct 1192 Lys AsnMet Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pro 315 320 325 ggggac aat gac atc ttt gag tcc ctg tac tac agg aag atg tac gga 1240 Gly AspAsn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly 330 335 340 345gac atg gcg taa agccaggaag taagggacac gaactcatta gactataact 1292 Asp MetAla * tgaactgagt tgcatctcat tttcttctgt aaaaacaatt acagtagcac attaatttaa1352 atctgtgttt ttaactaccg tgggaggaac tatcccacca aagtgagaac gttatgtcat1412 ggccatacaa gtagtctgtc aacctcagac actggtttcg agacagttta cacttgacag1472 ttgttcatta gcgcacagtg ccagaacgca cactgaggtg agtctcctgg aacagtggag1532 atgccaggag aaagaaagac aggtactagc tgaggttatt ttaaaagcag cagtgtgcct1592 actttttgga gtgtaaccgg ggagggcaat tatagcatgc ttgcagacag acctgctcta1652 gcgagagctg agcatgtgtc ctccactaga tgaggctgag tccagctgtt ctttaagaac1712 agcagtttca gctctgacca ttctgattcc agtgacactt gtcaggagtc agagccttgt1772 ctgttagact ggacagcttg tggcaagtaa gtttgcctgt aacaagccag atttttattg1832 atattgtaaa tattgtggat atatatatat atatatttgt acagttatct aagttaattt1892 aaagtcattt gtttttgttt taagtgcttt tgggatttta aactgatagc ctcaaactcc1952 aaacaccata ggtaggacac gaagcttatc tgtgattcaa aacaaaggag atactgcagt2012 gggaattgtg acctgagtga ctctctgtca gaacaaatgc tgtgcaggtg ataaagctat2072 gtattggaag tcagatttct agtaggaaat gtggtcaaat ccctgttggt gaacaaatgg2132 cctttattaa gaaatggctg gctcagggta aggtccgatt cctaccagga agtgcttgct2192 gcttctttga ttatgactgg tttggggtgg ggggcagttt atttgttgag agtgtgacca2252 aaagttacat gtttgcacct ttctagttga aaataaagta tatatatatt ttttatatga2312 aaaaaaaaaa aaaaaaaaaa aa 2334 11 21 DNA Artificial Sequence PCRPrimer 11 gctcagggta aggtccgatt c 21 12 15 DNA Artificial Sequence PCRPrimer 12 gccccccacc ccaaa 15 13 31 DNA Artificial Sequence PCR Probe 13tcataatcaa agaagcagca agcacttcct g 31 14 20 DNA Artificial Sequence PCRPrimer 14 ggcaaattca acggcacagt 20 15 20 DNA Artificial Sequence PCRPrimer 15 gggtctcgct cctggaagat 20 16 27 DNA Artificial Sequence PCRProbe 16 aaggccgaga atgggaagct tgtcatc 27 17 2312 DNA Homo sapiens CDS(146)...(1195) 17 tccagtgacg gagccgcccg gccgacagcc ccgagacgac agcccggcgcgtcccggtcc 60 ccacctccga ccaccgccag cgctccaggc cccgcgctcc ccgctcgccgccaccgcgcc 120 ctccgctccg cccgcagtgc caacc atg acc gcc gcc agt atg ggcccc gtc 172 Met Thr Ala Ala Ser Met Gly Pro Val 1 5 cgc gtc gcc ttc gtggtc ctc ctc gcc ctc tgc agc cgg ccg gcc gtc 220 Arg Val Ala Phe Val ValLeu Leu Ala Leu Cys Ser Arg Pro Ala Val 10 15 20 25 ggc cag aac tgc agcggg ccg tgc cgg tgc ccg gac gag ccg gcg ccg 268 Gly Gln Asn Cys Ser GlyPro Cys Arg Cys Pro Asp Glu Pro Ala Pro 30 35 40 cgc tgc ccg gcg ggc gtgagc ctc gtg ctg gac ggc tgc ggc tgc tgc 316 Arg Cys Pro Ala Gly Val SerLeu Val Leu Asp Gly Cys Gly Cys Cys 45 50 55 cgc gtc tgc gcc aag cag ctgggc gag ctg tgc acc gag cgc gac ccc 364 Arg Val Cys Ala Lys Gln Leu GlyGlu Leu Cys Thr Glu Arg Asp Pro 60 65 70 tgc gac ccg cac aag ggc ctc ttctgt gac ttc ggc tcc ccg gcc aac 412 Cys Asp Pro His Lys Gly Leu Phe CysAsp Phe Gly Ser Pro Ala Asn 75 80 85 cgc aag atc ggc gtg tgc acc gcc aaagat ggt gct ccc tgc atc ttc 460 Arg Lys Ile Gly Val Cys Thr Ala Lys AspGly Ala Pro Cys Ile Phe 90 95 100 105 ggt ggt acg gtg tac cgc agc ggagag tcc ttc cag agc agc tgc aag 508 Gly Gly Thr Val Tyr Arg Ser Gly GluSer Phe Gln Ser Ser Cys Lys 110 115 120 tac cag tgc acg tgc ctg gac ggggcg gtg ggc tgc atg ccc ctg tgc 556 Tyr Gln Cys Thr Cys Leu Asp Gly AlaVal Gly Cys Met Pro Leu Cys 125 130 135 agc atg gac gtt cgt ctg ccc agccct gac tgc ccc ttc ccg agg agg 604 Ser Met Asp Val Arg Leu Pro Ser ProAsp Cys Pro Phe Pro Arg Arg 140 145 150 gtc aag ctg ccc ggg aaa tgc tgcgag gag tgg gtg tgt gac gag ccc 652 Val Lys Leu Pro Gly Lys Cys Cys GluGlu Trp Val Cys Asp Glu Pro 155 160 165 aag gac caa acc gtg gtt ggg cctgcc ctc gcg gct tac cga ctg gaa 700 Lys Asp Gln Thr Val Val Gly Pro AlaLeu Ala Ala Tyr Arg Leu Glu 170 175 180 185 gac acg ttt ggc cca gac ccaact atg att aga gcc aac tgc ctg gtc 748 Asp Thr Phe Gly Pro Asp Pro ThrMet Ile Arg Ala Asn Cys Leu Val 190 195 200 cag acc aca gag tgg agc gcctgt tcc aag acc tgt ggg atg ggc atc 796 Gln Thr Thr Glu Trp Ser Ala CysSer Lys Thr Cys Gly Met Gly Ile 205 210 215 tcc acc cgg gtt acc aat gacaac gcc tcc tgc agg cta gag aag cag 844 Ser Thr Arg Val Thr Asn Asp AsnAla Ser Cys Arg Leu Glu Lys Gln 220 225 230 agc cgc ctg tgc atg gtc aggcct tgc gaa gct gac ctg gaa gag aac 892 Ser Arg Leu Cys Met Val Arg ProCys Glu Ala Asp Leu Glu Glu Asn 235 240 245 att aag aag ggc aaa aag tgcatc cgt act ccc aaa atc tcc aag cct 940 Ile Lys Lys Gly Lys Lys Cys IleArg Thr Pro Lys Ile Ser Lys Pro 250 255 260 265 atc aag ttt gag ctt tctggc tgc acc agc atg aag aca tac cga gct 988 Ile Lys Phe Glu Leu Ser GlyCys Thr Ser Met Lys Thr Tyr Arg Ala 270 275 280 aaa ttc tgt gga gta tgtacc gac ggc cga tgc tgc acc ccc cac aga 1036 Lys Phe Cys Gly Val Cys ThrAsp Gly Arg Cys Cys Thr Pro His Arg 285 290 295 acc acc acc ctg ccg gtggag ttc aag tgc cct gac ggc gag gtc atg 1084 Thr Thr Thr Leu Pro Val GluPhe Lys Cys Pro Asp Gly Glu Val Met 300 305 310 aag aag aac atg atg ttcatc aag acc tgt gcc tgc cat tac aac tgt 1132 Lys Lys Asn Met Met Phe IleLys Thr Cys Ala Cys His Tyr Asn Cys 315 320 325 ccc gga gac aat gac atcttt gaa tcg ctg tac tac agg aag atg tac 1180 Pro Gly Asp Asn Asp Ile PheGlu Ser Leu Tyr Tyr Arg Lys Met Tyr 330 335 340 345 gga gac atg gca tgaagccagagag tgagagacat taactcatta gactggaact 1235 Gly Asp Met Ala *tgaactgatt cacatctcat ttttccgtaa aaatgatttc agtagcacaa gttatttaaa 1295tctgtttttc taactggggg aaaagattcc cacccaattc aaaacattgt gccatgtcaa 1355acaaatagtc tatcttcccc agacactggt ttgaagaatg ttaagacttg acagtggaac 1415tacattagta cacagcacca gaatgtatat taaggtgtgg ctttaggagc agtgggaggg 1475taccagcaga aaggttagta tcatcagata gctcttatac gagtaatatg cctgctattt 1535gaagtgtaat tgagaaggaa aattttagcg tgctcactga cctgcctgta gccccagtga 1595cagctaggat gtgcattctc cagccatcaa gagactgagt caagttgttc cttaagtcag 1655aacagcagac tcagctctga cattctgatt cgaatgacac tgttcaggaa tcggaatcct 1715gtcgattaga ctggacagct tgtggcaagt gaatttcctg taacaagcca gattttttaa 1775aatttatatt gtaaatattg tgtgtgtgtg tgtgtgtgta tatatatata tatatgtaca 1835gttatctaag ttaatttaaa gttgtttgtg cctttttatt tttgttttta atgctttgat 1895atttcaatgt tagcctcaat ttctgaacac cataggtaga atgtaaagct tgtctgatcg 1955ttcaaagcat gaaatggata cttatatgga aattctctca gatagaatga cagtccgtca 2015aaacagattg tttgcaaagg ggaggcatca gtgtccttgg caggctgatt tctaggtagg 2075aaatgtggta gctcacgctc acttttaatg aacaaatggc ctttattaaa aactgagtga 2135ctctatatag ctgatcagtt ttttcacctg gaagcatttg tttctacttt gatatgactg 2195tttttcggac agtttatttg ttgagagtgt gaccaaaagt tacatgtttg cacctttcta 2255gttgaaaata aagtatattt tttctaaaaa aaaaaaaaaa cgacagcaac ggaattc 2312 182078 DNA Homo sapiens CDS (131)...(1180) 18 cccggccgac agccccgagacgacagcccg gcgcgtcccg gtccccacct ccgaccaccg 60 ccagcgctcc aggccccgccgctccccgct cgccgccacc gcgccctccg ctccgcccgc 120 agtgccaacc atg acc gccgcc agt atg ggc ccc gtc cgc gtc gcc ttc 169 Met Thr Ala Ala Ser Met GlyPro Val Arg Val Ala Phe 1 5 10 gtg gtc ctc ctc gcc ctc tgc agc cgg ccggcc gtc ggc cag aac tgc 217 Val Val Leu Leu Ala Leu Cys Ser Arg Pro AlaVal Gly Gln Asn Cys 15 20 25 agc ggg ccg tgc cgg tgc ccg gac gag ccg gcgccg cgc tgc ccg gcg 265 Ser Gly Pro Cys Arg Cys Pro Asp Glu Pro Ala ProArg Cys Pro Ala 30 35 40 45 ggc gtg agc ctc gtg ctg gac ggc tgc ggc tgctgc cgc gtc tgc gcc 313 Gly Val Ser Leu Val Leu Asp Gly Cys Gly Cys CysArg Val Cys Ala 50 55 60 aag cag ctg ggc gag ctg tgc acc gag cgc gac ccatgc gac ccg cac 361 Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro CysAsp Pro His 65 70 75 aag ggc cta ttc tgt cac ttc ggc tcc ccg gcc aac cgcaag atc ggc 409 Lys Gly Leu Phe Cys His Phe Gly Ser Pro Ala Asn Arg LysIle Gly 80 85 90 gtg tgc acc gcc aaa gat ggt gct ccc tgc atc ttc ggt ggtacg gtg 457 Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly ThrVal 95 100 105 tac cgc agc gga gag tcc ttc cag agc agc tgc aag tac cagtgc acg 505 Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln CysThr 110 115 120 125 tgc ctg gac ggg gcg gtg ggc tgc atg ccc ctg tgc agcatg gac gtt 553 Cys Leu Asp Gly Ala Val Gly Cys Met Pro Leu Cys Ser MetAsp Val 130 135 140 cgt ctg ccc agc cct gac tgc ccc ttc ccg agg agg gtcaag ctg ccc 601 Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val LysLeu Pro 145 150 155 ggg aaa tgc tgc gag gag tgg gtg tgt gac gag ccc aaggac caa acc 649 Gly Lys Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Lys AspGln Thr 160 165 170 gtg gtt ggg cct gcc ctc gcg gct tac cga ctg gaa gacacg ttt ggc 697 Val Val Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp ThrPhe Gly 175 180 185 cca gac cca act atg att aga gcc aac tgc ctg gtc cagacc aca gag 745 Pro Asp Pro Thr Met Ile Arg Ala Asn Cys Leu Val Gln ThrThr Glu 190 195 200 205 tgg agc gcc tgt tcc aag acc tgt ggg atg ggc atctcc acc cgg gtt 793 Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile SerThr Arg Val 210 215 220 acc aat gac aac gcc tcc tgc agg cta gag aag cagagc cgc ctg tgc 841 Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln SerArg Leu Cys 225 230 235 atg gtc agg cct tgc gaa gct gac ctg gaa gag aacatt aag aag ggc 889 Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn IleLys Lys Gly 240 245 250 aaa aag tgc atc cgt act ccc aaa atc tcc aag cctatc aag ttt gag 937 Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro IleLys Phe Glu 255 260 265 ctt tct ggc tgc acc agc atg aag aca tac cga gctaaa ttc tgt gga 985 Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala LysPhe Cys Gly 270 275 280 285 gta tgt acc gac ggc cga tgc tgc acc ccc cacaga acc acc acc ctg 1033 Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His ArgThr Thr Thr Leu 290 295 300 ccg gtg gag ttc aag tgc cct gac ggc gag gtcatg aag aag aac atg 1081 Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Val MetLys Lys Asn Met 305 310 315 atg ttc atc aag acc tgt gcc tgc cat tac aactgt ccc gga gac aat 1129 Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn CysPro Gly Asp Asn 320 325 330 gac atc ttt gaa tcg ctg tac tac agg aag atgtac gga gac atg gca 1177 Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met TyrGly Asp Met Ala 335 340 345 tga agccagagag tgagagacat taactcattagactggaact tgaactgatt 1230 cacatctcat ttttccgtaa aaatgatttc agtagcacaagttatttaaa tctgtttttc 1290 taactggggg aaaagattcc cacccaattc aaaacattgtgccatgtcaa acaaatagtc 1350 tatcaacccc agacactggt ttgaagaatg ttaagacttgacagtggaac tacattagta 1410 cacagcacca gaatgtatat taaggtgtgg ctttaggagcagtgggaggg taccagcaga 1470 aaggttagta tcatcagata gcatcttata cgagtaatatgcctgctatt tgaagtgtaa 1530 ttgagaagga aaattttagc gtgctcactg acctgcctgtagccccagtg acagctagga 1590 tgtgcattct ccagccatca agagactgag tcaagttgttccttaagtca gaacagcaga 1650 ctcagctctg acattctgat tcgaatgaca ctgttcaggaatcggaatcc tgtcgattag 1710 actggacagc ttgtggcaag tgaatttgcc tgtaacaagccagatttttt aaaatttata 1770 ttgtaaatat tgtgtgtgtg tgtgtgtgtg tatatatatatatatgtaca gttatctaag 1830 ttaatttaaa gttgtttgtg cctttttatt tttgtttttaatgctttgat atttcaatgt 1890 tagcctcaat ttctgaacac cataggtaga atgtaaagcttgtctgatcg ttcaaagcat 1950 gaaatggata cttatatgga aattctgctc agatagaatgacagtccgtc aaaacagatt 2010 gtttgcaaag gggaggcatc agtgtccttg gcaggctgatttctaggtag gaaatgtggt 2070 agcctcac 2078 19 2280 DNA Homo sapiens CDS(143)...(1192) 19 gacggcagcc gccccggccg acagccccga gacgacagcc cggcgcgtcccggtccccac 60 ctccgaccac cgccagcgct ccaggccccg ccgctccccg ctcgccgccaccgcgccctc 120 cgctccgccc gcagtgccaa cc atg acc gcc gcc agt atg ggc cccgtc cgc 172 Met Thr Ala Ala Ser Met Gly Pro Val Arg 1 5 10 gtc gcc ttcgtg gtc ctc ctc gcc ctc tgc agc cgg ccg gcc gtc ggc 220 Val Ala Phe ValVal Leu Leu Ala Leu Cys Ser Arg Pro Ala Val Gly 15 20 25 cag aac tgc agcggg ccg tgc cgg tgc ccg gac gag ccg gcg ccg cgc 268 Gln Asn Cys Ser GlyPro Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg 30 35 40 tgc ccg gcg ggc gtgagc ctc gtg ctg gac ggc tgc ggc tgc tgc cgc 316 Cys Pro Ala Gly Val SerLeu Val Leu Asp Gly Cys Gly Cys Cys Arg 45 50 55 gtc tgc gcc aag cag ctgggc gag ctg tgc acc gag cgc gac cca tgc 364 Val Cys Ala Lys Gln Leu GlyGlu Leu Cys Thr Glu Arg Asp Pro Cys 60 65 70 gac ccg cac aag ggc cta ttctgt cac ttc ggc tcc ccg gcc aac cgc 412 Asp Pro His Lys Gly Leu Phe CysHis Phe Gly Ser Pro Ala Asn Arg 75 80 85 90 aag atc ggc gtg tgc acc gccaaa gat ggt gct ccc tgc atc ttc ggt 460 Lys Ile Gly Val Cys Thr Ala LysAsp Gly Ala Pro Cys Ile Phe Gly 95 100 105 ggt acg gtg tac cgc agc ggagag tcc ttc cag agc agc tgc aag tac 508 Gly Thr Val Tyr Arg Ser Gly GluSer Phe Gln Ser Ser Cys Lys Tyr 110 115 120 cag tgc acg tgc ctg gac ggggcg gtg ggc tgc atg ccc ctg tgc agc 556 Gln Cys Thr Cys Leu Asp Gly AlaVal Gly Cys Met Pro Leu Cys Ser 125 130 135 atg gac gtt cgt ctg ccc agccct gac tgc ccc ttc ccg agg agg gtc 604 Met Asp Val Arg Leu Pro Ser ProAsp Cys Pro Phe Pro Arg Arg Val 140 145 150 aag ctg ccc ggg aaa tgc tgcgag gag tgg gtg tgt gac gag ccc aag 652 Lys Leu Pro Gly Lys Cys Cys GluGlu Trp Val Cys Asp Glu Pro Lys 155 160 165 170 gac caa acc gtg gtt gggcct gcc ctc gcg gct tac cga ctg gaa gac 700 Asp Gln Thr Val Val Gly ProAla Leu Ala Ala Tyr Arg Leu Glu Asp 175 180 185 acg ttt ggc cca gac ccaact atg att aga gcc aac tgc ctg gtc cag 748 Thr Phe Gly Pro Asp Pro ThrMet Ile Arg Ala Asn Cys Leu Val Gln 190 195 200 acc aca gag tgg agc gcctgt tcc aag acc tgt ggg atg ggc atc tcc 796 Thr Thr Glu Trp Ser Ala CysSer Lys Thr Cys Gly Met Gly Ile Ser 205 210 215 acc cgg gtt acc aat gacaac gcc tcc tgc agg cta gag aag cag agc 844 Thr Arg Val Thr Asn Asp AsnAla Ser Cys Arg Leu Glu Lys Gln Ser 220 225 230 cgc ctg tgc atg gtc aggcct tgc gaa gct gac ctg gaa gag aac att 892 Arg Leu Cys Met Val Arg ProCys Glu Ala Asp Leu Glu Glu Asn Ile 235 240 245 250 aag aag ggc aaa aagtgc atc cgt act ccc aaa atc tcc aag cct atc 940 Lys Lys Gly Lys Lys CysIle Arg Thr Pro Lys Ile Ser Lys Pro Ile 255 260 265 aag ttt gag ctt tctggc tgc acc agc atg aag aca tac cga gct aaa 988 Lys Phe Glu Leu Ser GlyCys Thr Ser Met Lys Thr Tyr Arg Ala Lys 270 275 280 ttc tgt gga gta tgtacc gac ggc cga tgc tgc acc ccc cac aga acc 1036 Phe Cys Gly Val Cys ThrAsp Gly Arg Cys Cys Thr Pro His Arg Thr 285 290 295 acc acc ctg ccg gtggag ttc aag tgc cct gac ggc gag gtc atg aag 1084 Thr Thr Leu Pro Val GluPhe Lys Cys Pro Asp Gly Glu Val Met Lys 300 305 310 aag aac atg atg ttcatc aag acc tgt gcc tgc cat tac aac tgt ccc 1132 Lys Asn Met Met Phe IleLys Thr Cys Ala Cys His Tyr Asn Cys Pro 315 320 325 330 gga gac aat gacatc ttt gaa tcg ctg tac tac agg aag atg tac gga 1180 Gly Asp Asn Asp IlePhe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly 335 340 345 gac atg gca tgaagccagagag tgagagacat taactcatta gactggaact 1232 Asp Met Ala *tgaactgatt cacatctcat ttttccgtaa aaatgatttc agtagcacaa gttatttaaa 1292tctgtttttc taactggggg aaaagattcc cacccaattc aaaacattgt gccatgtcaa 1352acaaatagtc tatcaacccc agacactggt ttgaagaatg ttaagacttg acagtggaac 1412tacattagta cacagcacca gaatgtatat taaggtgtgg ctttaggagc agtgggaggg 1472taccagcaga aaggttagta tcatcagata gcatcttata cgagtaatat gcctgctatt 1532tgaagtgtaa ttgagaagga aaattttagc gtgctcactg acctgcctgt agccccagtg 1592acagctagga tgtgcattct ccagccatca agagactgag tcaagttgtt ccttaagtca 1652gaacagcaga ctcagctctg acattctgat tcgaatgaca ctgttcagga atcggaatcc 1712tgtcgattag actggacagc ttgtggcaag tgaatttgcc tgtaacaagc cagatttttt 1772aaaatttata ttgtaaatat tgtgtgtgtg tgtgtgtgtg tatatatata tatatgtaca 1832gttatctaag ttaatttaaa gttgtttgtg cctttttatt tttgttttta atgctttgat 1892atttcaatgt tagcctcaat ttctgaacac cataggtaga atgtaaagct tgtctgatcg 1952ttcaaagcat gaaatggata cttatatgga aattctgctc agatagaatg acagtccgtc 2012aaaacagatt gtttgcaaag gggaggcatc agtgtccttg gcaggctgat ttctaggtag 2072gaaatgtggt agcctcactt ttaatgaaca aatggccttt attaaaaact gagtgactct 2132atatagctga tcagtttttt cacctggaag catttgtttc tactttgata tgactgtttt 2192tcggacagtt tatttgttga gagtgtgacc aaaagttaca tgtttgcacc tttctagttg 2252aaaataaagt gtatattttt tctataaa 2280 20 20 DNA Artificial SequenceAntisense Oligonucleotide 20 gcagttggct ctaatcatag 20 21 20 DNAArtificial Sequence Antisense Oligonucleotide 21 tgaccatgca caggcggctc20 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22 ctcaaacttgataggcttgg 20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23tttagctcgg tatgtcttca 20 24 20 DNA Artificial Sequence AntisenseOligonucleotide 24 cttgaactcc accggcaggg 20 25 20 DNA ArtificialSequence Antisense Oligonucleotide 25 ggtcttgatg aacatcatgt 20 26 20 DNAArtificial Sequence Antisense Oligonucleotide 26 gacagttgta atggcaggca20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27 ccgtacatcttcctgtagta 20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28ccagctgctt ggcgcagacg 20 29 20 DNA Artificial Sequence AntisenseOligonucleotide 29 tctggaccag gcagttggct 20 30 20 DNA ArtificialSequence Antisense Oligonucleotide 30 tgtggtctgg accaggcagt 20 31 20 DNAArtificial Sequence Antisense Oligonucleotide 31 cactctgtgg tctggaccag20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32 gatgcactttttgcccttct 20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33gccagaaagc tcaaacttga 20 34 20 DNA Artificial Sequence AntisenseOligonucleotide 34 gtgcagccag aaagctcaaa 20 35 20 DNA ArtificialSequence Antisense Oligonucleotide 35 caggtcttga tgaacatcat 20 36 20 DNAArtificial Sequence Antisense Oligonucleotide 36 aggcacaggt cttgatgaac20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37 atggcaggcacaggtcttga 20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38acagttgtaa tggcaggcac 20 39 20 DNA Artificial Sequence AntisenseOligonucleotide 39 ccacaagctg tccagtctaa 20 40 20 DNA ArtificialSequence Antisense Oligonucleotide 40 acttgccaca agctgtccag 20 41 20 DNAArtificial Sequence Antisense Oligonucleotide 41 ttaacttaga taactgtaca20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42 ttaaattaacttagataact 20 43 20 DNA Artificial Sequence Antisense Oligonucleotide 43ttaataaagg ccatttgttc 20 44 20 DNA Artificial Sequence AntisenseOligonucleotide 44 cactctcaac aaataaactg 20 45 20 DNA ArtificialSequence Antisense Oligonucleotide 45 ggtcacactc tcaacaaata 20 46 20 DNAArtificial Sequence Antisense Oligonucleotide 46 cttttggtca cactctcaac20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47 tgtaacttttggtcacactc 20 48 20 DNA Artificial Sequence Antisense Oligonucleotide 48aaacatgtaa cttttggtca 20 49 20 DNA Artificial Sequence AntisenseOligonucleotide 49 ctttattttc aactagaaag 20 50 20 DNA ArtificialSequence Antisense Oligonucleotide 50 cagctgcttg gcgcagacgc 20 51 20 DNAArtificial Sequence Antisense Oligonucleotide 51 ccttgggctc gtcacacacc20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52 tctgtggtctggaccaggca 20 53 20 DNA Artificial Sequence Antisense Oligonucleotide 53cagccagaaa gctcaaactt 20 54 20 DNA Artificial Sequence AntisenseOligonucleotide 54 ctggtgcagc cagaaagctc 20 55 20 DNA ArtificialSequence Antisense Oligonucleotide 55 acaggtcttg atgaacatca 20 56 20 DNAArtificial Sequence Antisense Oligonucleotide 56 gcaggcacag gtcttgatga20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57 taatggcaggcacaggtctt 20 58 20 DNA Artificial Sequence Antisense Oligonucleotide 58ccatgtctcc gtacatcttc 20 59 20 DNA Artificial Sequence AntisenseOligonucleotide 59 cttgccacaa gctgtccagt 20 60 20 DNA ArtificialSequence Antisense Oligonucleotide 60 aaaaatctgg cttgttacag 20 61 20 DNAArtificial Sequence Antisense Oligonucleotide 61 ctttaaatta acttagataa20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62 tttggtcacactctcaacaa 20 63 20 DNA Artificial Sequence Antisense Oligonucleotide 63gtaacttttg gtcacactct 20 64 20 DNA Artificial Sequence AntisenseOligonucleotide 64 caaacatgta acttttggtc 20 65 20 DNA ArtificialSequence Antisense Oligonucleotide 65 actttatttt caactagaaa 20 66 20 DNAArtificial Sequence Antisense Oligonucleotide 66 cggcggtcat ggttggcact20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67 cccatactggcggcggtcat 20 68 20 DNA Artificial Sequence Antisense Oligonucleotide 68ccgtccagca cgaggctcac 20 69 20 DNA Artificial Sequence AntisenseOligonucleotide 69 agaggccctt gtgcgggtcg 20 70 20 DNA ArtificialSequence Antisense Oligonucleotide 70 gagccgaagt cacagaagag 20 71 20 DNAArtificial Sequence Antisense Oligonucleotide 71 aaggactctc cgctgcggta20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72 cacgtgcactggtacttgca 20 73 20 DNA Artificial Sequence Antisense Oligonucleotide 73tcgcagcatt tcccgggcag 20 74 20 DNA Artificial Sequence AntisenseOligonucleotide 74 ctcctcgcag catttcccgg 20 75 20 DNA ArtificialSequence Antisense Oligonucleotide 75 gggctcgtca cacacccact 20 76 20 DNAArtificial Sequence Antisense Oligonucleotide 76 gtctgggcca aacgtgtctt20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77 tctaatcatagttgggtctg 20 78 20 DNA Artificial Sequence Antisense Oligonucleotide 78gaccaggcag ttggctctaa 20 79 20 DNA Artificial Sequence AntisenseOligonucleotide 79 ctctagcctg caggaggcgt 20 80 20 DNA ArtificialSequence Antisense Oligonucleotide 80 atgttctctt ccaggtcagc 20 81 20 DNAArtificial Sequence Antisense Oligonucleotide 81 ggagattttg ggagtacgga20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82 ttgataggcttggagatttt 20 83 20 DNA Artificial Sequence Antisense Oligonucleotide 83cacagaattt agctcggtat 20 84 20 DNA Artificial Sequence AntisenseOligonucleotide 84 ggccgtcggt acatactcca 20 85 20 DNA ArtificialSequence Antisense Oligonucleotide 85 tccaccggca gggtggtggt 20 86 20 DNAArtificial Sequence Antisense Oligonucleotide 86 cagggcactt gaactccacc20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87 ccgtcagggcacttgaactc 20 88 20 DNA Artificial Sequence Antisense Oligonucleotide 88ggacagttgt aatggcaggc 20 89 20 DNA Artificial Sequence AntisenseOligonucleotide 89 gtagtacagc gattcaaaga 20 90 20 DNA ArtificialSequence Antisense Oligonucleotide 90 tctggcttca tgccatgtct 20 91 20 DNAArtificial Sequence Antisense Oligonucleotide 91 tctctcactc tctggcttca20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92 tacggaaaaatgagatgtga 20 93 20 DNA Artificial Sequence Antisense Oligonucleotide 93atttaaataa cttgtgctac 20 94 20 DNA Artificial Sequence AntisenseOligonucleotide 94 ttcttcaaac cagtgtctgg 20 95 20 DNA ArtificialSequence Antisense Oligonucleotide 95 cagtgagcac gctaaaattt 20 96 20 DNAArtificial Sequence Antisense Oligonucleotide 96 gttctgactt aaggaacaac20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97 gctgtccagtctaatcgaca 20 98 2330 DNA Mus musculus CDS (204)...(1250) 98 agactcagccagatccactc cagctccgac cccaggagac cgacctcctc cagacggcag 60 cagccccagcccagccgaca accccagacg ccaccgcctg gagcgtccag acaccaacct 120 ccgcccctgtccgaatccag gctccagccg cgcctctcgt cgcctctgca ccctgctgtg 180 catcctcctaccgcgtcccg atc atg ctc gcc tcc gtc gca ggt ccc atc agc 233 Met Leu AlaSer Val Ala Gly Pro Ile Ser 1 5 10 ctc gcc ttg gtg ctc ctc gcc ctc tgcacc cgg cct gct acg ggc cag 281 Leu Ala Leu Val Leu Leu Ala Leu Cys ThrArg Pro Ala Thr Gly Gln 15 20 25 gac tgc agc gcg caa tgt cag tgc gca gccgaa gca gcg ccg cac tgc 329 Asp Cys Ser Ala Gln Cys Gln Cys Ala Ala GluAla Ala Pro His Cys 30 35 40 ccc gcc ggc gtg agc ctg gtg ctg gac ggc tgcggc tgc tgc cgc gtc 377 Pro Ala Gly Val Ser Leu Val Leu Asp Gly Cys GlyCys Cys Arg Val 45 50 55 tgc gcc aag cag ctg gga gaa ctg tgt acg gag cgtgac ccc tgc gac 425 Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg AspPro Cys Asp 60 65 70 cca cac aag ggc ctc ttc tgc gat ttc ggc tcc ccc gccaac cgc aag 473 Pro His Lys Gly Leu Phe Cys Asp Phe Gly Ser Pro Ala AsnArg Lys 75 80 85 90 att gga gtg tgc act gcc aaa gat ggt gca ccc tgt gtcttc ggt ggg 521 Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Val PheGly Gly 95 100 105 tcg gtg tac cgc agc ggt gag tcc ttc caa agc agc tgcaaa tac caa 569 Ser Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys LysTyr Gln 110 115 120 tgc act tgc ctg gat ggg gcc gtg ggc tgc gtg ccc ctatgc agc atg 617 Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Val Pro Leu CysSer Met 125 130 135 gac gtg cgc ctg ccc agc cct gac tgc ccc ttc ccg agaagg gtc aag 665 Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg ArgVal Lys 140 145 150 ctg cct ggg aaa tgc tgc gag gag tgg gtg tgt gac gagccc aag gac 713 Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp Glu ProLys Asp 155 160 165 170 cgc aca gca gtt ggc cct gcc cta gct gcc tac cgactg gaa gac aca 761 Arg Thr Ala Val Gly Pro Ala Leu Ala Ala Tyr Arg LeuGlu Asp Thr 175 180 185 ttt ggc cca gac cca act atg atg cga gcc aac tgcctg gtc cag acc 809 Phe Gly Pro Asp Pro Thr Met Met Arg Ala Asn Cys LeuVal Gln Thr 190 195 200 aca gag tgg agc gcc tgt tct aag acc tgt gga atgggc atc tcc acc 857 Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met GlyIle Ser Thr 205 210 215 cga gtt acc aat gac aat acc ttc tgc aga ctg gagaag cag agc cgc 905 Arg Val Thr Asn Asp Asn Thr Phe Cys Arg Leu Glu LysGln Ser Arg 220 225 230 ctc tgc atg gtc agg ccc tgc gaa gct gac ctg gaggaa aac att aag 953 Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu GluAsn Ile Lys 235 240 245 250 aag ggc aaa aag tgc atc cgg aca cct aaa atcgcc aag cct gtc aag 1001 Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile AlaLys Pro Val Lys 255 260 265 ttt gag ctt tct ggc tgc acc agt gtg aag acatac agg gct aag ttc 1049 Phe Glu Leu Ser Gly Cys Thr Ser Val Lys Thr TyrArg Ala Lys Phe 270 275 280 tgc ggg gtg tgc aca gac ggc cgc tgc tgc acaccg cac aga acc acc 1097 Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr ProHis Arg Thr Thr 285 290 295 act ctg cca gtg gag ttc aaa tgc ccc gat ggcgag atc atg aaa aag 1145 Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly GluIle Met Lys Lys 300 305 310 aat atg atg ttc atc aag acc tgt gcc tgc cattac aac tgt cct ggg 1193 Asn Met Met Phe Ile Lys Thr Cys Ala Cys His TyrAsn Cys Pro Gly 315 320 325 330 gac aat gac atc ttt gag tcc ctg tac tacagg aag atg tac gga gac 1241 Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr ArgLys Met Tyr Gly Asp 335 340 345 atg gcg taa agccaggaag taagggacacgaactcatta gactataact 1290 Met Ala * tgaactgagt tgcatctcat tttcttctgtaaaaacaatt acagtagcac attaatttaa 1350 atctgtgttt ttaactaccg tgggaggaactatcccacca aagtgagaac gttatgtcat 1410 ggccatacaa gtagtctgtc aacctcagacactggtttcg agacagttta cacttgacag 1470 ttgttcatta gcgcacagtg ccagaacgcacactgaggtg agtctcctgg aacagtggag 1530 atgccaggag aaagaaagac aggtactagctgaggttatt ttaaaagcag cagtgtgcct 1590 actttttgga gtgtaaccgg ggagggaaattatagcatgc ttgcagacag acctgctcta 1650 gcgagagctg agcatgtgtc ctccactagatgaggctgag tccagctgtt ctttaagaac 1710 agcagtttca gctctgacca ttctgattccagtgacactt gtcaggagtc agagccttgt 1770 ctgttagact ggacagcttg tggcaagtaagtttgcctgt aacaagccag atttttattg 1830 atattgtaaa tattgtggat atatatatatatatatatat atttgtacag ttatctaagt 1890 taatttaaag tcatttgttt ttgttttaagtgcttttggg attttaaact gatagcctca 1950 aactccaaac accataggta ggacacgaagcttatctgtg attcaaaaca aaggagatac 2010 tgcagtggga attgtgacct gagtgactctctgtcagaac aaacaaatgc tgtgcaggtg 2070 ataaagctat gtattggaag tcagatttctagtaggaaat gtggtcaaat ccctgttggt 2130 gaacaaatgg cctttattaa gaaatggctggctcagggta aggtccgatt cctaccagga 2190 agtgcttgct gcttctttga ttatgactggtttggggtgg ggggcagttt atttgttgag 2250 agtgtgacca aaagttacat gtttgcactttctagttgaa aataaagtat atatatattt 2310 ttatatgaaa aaaaaaaaaa 2330 99 20DNA Artificial Sequence Antisense Oligonucleotide 99 actttttgcccttcttaatg 20 100 20 DNA Artificial Sequence Antisense Oligonucleotide100 gacgctccag gcggtggcgt 20 101 20 DNA Artificial Sequence AntisenseOligonucleotide 101 gtctggacgc tccaggcggt 20 102 20 DNA ArtificialSequence Antisense Oligonucleotide 102 cggctggagc ctggattcgg 20 103 20DNA Artificial Sequence Antisense Oligonucleotide 103 gagaggcgcggctggagcct 20 104 20 DNA Artificial Sequence Antisense Oligonucleotide104 acgcggtagg aggatgcaca 20 105 20 DNA Artificial Sequence AntisenseOligonucleotide 105 gaggcgagca tgatcgggac 20 106 20 DNA ArtificialSequence Antisense Oligonucleotide 106 ggcgcagacg cggcagcagc 20 107 20DNA Artificial Sequence Antisense Oligonucleotide 107 tgcttggcgcagacgcggca 20 108 20 DNA Artificial Sequence Antisense Oligonucleotide108 gcccttgtgt gggtcgcagg 20 109 20 DNA Artificial Sequence AntisenseOligonucleotide 109 aatcgcagaa gaggcccttg 20 110 20 DNA ArtificialSequence Antisense Oligonucleotide 110 accgacccac cgaagacaca 20 111 20DNA Artificial Sequence Antisense Oligonucleotide 111 ttggtatttgcagctgcttt 20 112 20 DNA Artificial Sequence Antisense Oligonucleotide112 cacgcagccc acggccccat 20 113 20 DNA Artificial Sequence AntisenseOligonucleotide 113 gcacgtccat gctgcatagg 20 114 20 DNA ArtificialSequence Antisense Oligonucleotide 114 gcagcttgac ccttctcggg 20 115 20DNA Artificial Sequence Antisense Oligonucleotide 115 actgctgtgcggtccttggg 20 116 20 DNA Artificial Sequence Antisense Oligonucleotide116 gtgtcttcca gtcggtaggc 20 117 20 DNA Artificial Sequence AntisenseOligonucleotide 117 aaggtattgt cattggtaac 20 118 20 DNA ArtificialSequence Antisense Oligonucleotide 118 ggctctgctt ctccagtctg 20 119 20DNA Artificial Sequence Antisense Oligonucleotide 119 tcttcacactggtgcagcca 20 120 20 DNA Artificial Sequence Antisense Oligonucleotide120 cgtctgtgca caccccgcag 20 121 20 DNA Artificial Sequence AntisenseOligonucleotide 121 cggtgtgcag cagcggccgt 20 122 20 DNA ArtificialSequence Antisense Oligonucleotide 122 tccactggca gagtggtggt 20 123 20DNA Artificial Sequence Antisense Oligonucleotide 123 catcatattctttttcatga 20 124 20 DNA Artificial Sequence Antisense Oligonucleotide124 gtcattgtcc ccaggacagt 20 125 20 DNA Artificial Sequence AntisenseOligonucleotide 125 tcctggcttt acgccatgtc 20 126 20 DNA ArtificialSequence Antisense Oligonucleotide 126 aaatgagatg caactcagtt 20 127 20DNA Artificial Sequence Antisense Oligonucleotide 127 tcagtgtgcgttctggcact 20 128 20 DNA Artificial Sequence Antisense Oligonucleotide128 gttccaggag actcacctca 20 129 20 DNA Artificial Sequence AntisenseOligonucleotide 129 tctccactgt tccaggagac 20 130 20 DNA ArtificialSequence Antisense Oligonucleotide 130 tctcctggca tctccactgt 20 131 20DNA Artificial Sequence Antisense Oligonucleotide 131 tttctttctcctggcatctc 20 132 20 DNA Artificial Sequence Antisense Oligonucleotide132 tccccggtta cactccaaaa 20 133 20 DNA Artificial Sequence AntisenseOligonucleotide 133 aggtctgtct gcaagcatgc 20 134 20 DNA ArtificialSequence Antisense Oligonucleotide 134 tgctcagctc tcgctagagc 20 135 20DNA Artificial Sequence Antisense Oligonucleotide 135 agtgtcactggaatcagaat 20 136 20 DNA Artificial Sequence Antisense Oligonucleotide136 caaatatata tatatatata 20 137 20 DNA Artificial Sequence AntisenseOligonucleotide 137 acttaaaaca aaaacaaatg 20 138 20 DNA ArtificialSequence Antisense Oligonucleotide 138 gctatcagtt taaaatccca 20 139 20DNA Artificial Sequence Antisense Oligonucleotide 139 gtgtcctacctatggtgttt 20 140 20 DNA Artificial Sequence Antisense Oligonucleotide140 tttgaatcac agataagctt 20 141 20 DNA Artificial Sequence AntisenseOligonucleotide 141 cagtatctcc tttgttttga 20 142 20 DNA ArtificialSequence Antisense Oligonucleotide 142 attcccactg cagtatctcc 20 143 20DNA Artificial Sequence Antisense Oligonucleotide 143 caggtcacaattcccactgc 20 144 20 DNA Artificial Sequence Antisense Oligonucleotide144 ctgacagaga gtcactcagg 20 145 20 DNA Artificial Sequence AntisenseOligonucleotide 145 gctttatcac ctgcacagca 20 146 20 DNA ArtificialSequence Antisense Oligonucleotide 146 tacatagctt tatcacctgc 20 147 20DNA Artificial Sequence Antisense Oligonucleotide 147 cttccaatacatagctttat 20 148 20 DNA Artificial Sequence Antisense Oligonucleotide148 tctgacttcc aatacatagc 20 149 20 DNA Artificial Sequence AntisenseOligonucleotide 149 atttgttcac caacagggat 20 150 20 DNA ArtificialSequence Antisense Oligonucleotide 150 ttaccctgag ccagccattt 20 151 20DNA Artificial Sequence Antisense Oligonucleotide 151 aagaagcagcaagcacttcc 20 152 20 DNA Artificial Sequence Antisense Oligonucleotide152 cagtcataat caaagaagca 20 153 20 DNA Artificial Sequence AntisenseOligonucleotide 153 atatacttta ttttcaacta 20

What is claimed is:
 1. A compound 8 to 50 nucleobases in length targetedto a nucleic acid molecule encoding connective tissue growth factor,wherein said compound specifically hybridizes with said nucleic acidmolecule encoding connective tissue growth factor and inhibits theexpression of connective tissue growth factor.
 2. The compound of claim1 which is an antisense oligonucleotide.
 3. The compound of claim 2wherein the antisense oligonucleotide has a sequence comprising SEQ IDNO: 24, 25, 27, 28, 33, 34, 35, 36, 38, 39, 41, 45, 46, 47, 48, 50, 52,58, 62, 63, 64, 68, 70, 72, 73, 81, 86, 88, 90, 91, 92, 95, 97, 30, 31,32, 37, 40, 42, 103, 104, 106, 108, 110, 111, 116, 117, 120, 122, 124,126, 127, 128, 130, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 151 or
 153. 4. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 5. The compound of claim 4 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 6. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 7. The compound of claim 6 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 9. The compound of claim 8 wherein the modifiednucleobase is a 5-methylcytosine.
 10. The compound of claim 2 whereinthe antisense oligonucleotide is a chimeric oligonucleotide.
 11. Acompound 8 to 50 nucleobases in length which specifically hybridizeswith at least an 8-nucleobase portion of an active site on a nucleicacid molecule encoding connective tissue growth factor.
 12. Acomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 13. The composition of claim 12 furthercomprising a colloidal dispersion system.
 14. The composition of claim12 wherein the compound is an antisense oligonucleotide.
 15. A method ofinhibiting the expression of connective tissue growth factor in cells ortissues comprising contacting said cells or tissues with the compound ofclaim 1 so that expression of connective tissue growth factor isinhibited.
 16. A method of treating an animal having a disease orcondition associated with connective tissue growth factor comprisingadministering to said animal a therapeutically or prophylacticallyeffective amount of the compound of claim 1 so that expression ofconnective tissue growth factor is inhibited.
 17. The method of claim 16wherein the disease or condition is a hyperproliferative disorder. 18.The method of claim 17 wherein the hyperproliferative disorder iscancer.
 19. The method of claim 18 wherein the cancer is selected fromthe group consisting of breast, prostate and renal cancer.
 20. Themethod of claim 16 wherein the disease or condition is selected from thegroup consisting of pulmonary fibrosis, renal fibrosis, scleroderma, andatherosclerosis.